Augmenting Moieties for Anti-Inflammatory Compounds

ABSTRACT

Augmented or synergized anti-inflammatory constructs are disclosed including anti-inflammatory amino acids covalently conjugated with other anti-inflammatory molecules such as nonsteroidal anti-inflammatory drugs, vanilloids and ketone bodies. Further conjugation with a choline bioisostere or an additional anti-inflammatory moiety further augments the anti-inflammatory activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 14/776,857, filed on Sep. 15, 2015, which is the U.S. NationalPhase of International Patent Application Serial No. PCT/US14/28329,filed on Mar. 14, 2014, which claims the benefit of priority under 35U.S.C. 119(e) of U.S. Provisional Application No. 61/790,870, filed onMar. 15, 2013, and of U.S. Provisional Application No. 61/793,842, filedon Mar. 15, 2013. The disclosures of all of the above are incorporatedherein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.U54AR055073 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to anti-inflammatory compounds which aresynergistically enhanced in their anti-inflammatory activity throughconjugation with specific amino acids and/or with specific otheranti-inflammatory components. Also disclosed are methods of increasingthe activity of an anti-inflammatory compound, which involve conjugatingthe anti-inflammatory compound with an amino acid and optionally furtherconjugating with a choline bioisostere, or conjugating one, two or moreanti-inflammatory compounds with each other, for example, terpene, aminoacid, vanilloid, or polyamine.

BACKGROUND OF THE INVENTION

The term “anti-inflammatory” refers to the property of a compound thatreduces inflammation. Anti-inflammatory drugs make up about half ofanalgesics, remedying pain by reducing inflammation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs thatprovide analgesic and antipyretic (fever-reducing) effects, and, inhigher doses, anti-inflammatory effects. The term “nonsteroidal”distinguishes these drugs from steroids, which, among a broad range ofother effects, have a similar eicosanoid-depressing, anti-inflammatoryaction. As analgesics, NSAIDs are unusual in that they are non-narcotic.The most prominent members of the NSAID group of drugs are aspirin,ibuprofen and naproxen.

The widespread use of NSAIDs has meant that the adverse effects of thesedrugs are well known and have become increasingly prevalent as thepopulation ages. The two main adverse drug reactions (ADRs) associatedwith NSAID use are gastrointestinal (GI) and renal effects. Theseeffects are dose-dependent and, in many cases, severe enough to pose therisk of ulcer perforation, upper gastrointestinal bleeding, and death,thereby limiting the use of NSAID therapy. An estimated 10-20% of NSAIDpatients experience dyspepsia, and NSAID-associated upper GI adverseevents are estimated to result in 103,000 hospitalizations and 16,500deaths per year in the United States and represent 43% of drug-relatedemergency visits. Thus, the clinical problems with NSAIDs and the needfor replacement anti-inflammatories are well recognized.

For at least these reasons, it would be desirable to find substitutesfor the current NSAIDs having increased anti-inflammatory potency and ahigher safety margin.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that one solution to this problem is toimprove the potency and safety of anti-inflammatory compounds throughthe covalent combination of component anti-inflammatory moieties and/orconjugation with a specific amino acid, optionally with furtherconjugation with a choline bioisostere.

Aspect I

Terpenes, amino acids, aliphatic polyamines such as spermine andspermidine, and vanilloid platforms (e.g., 4-hydroxy-3-methoxybenzylamine, commonly called vanillylamine; 4-hydroxy-3-methoxybenzyl alcohol,commonly called vanillyl alcohol; zingerone; [6]-paradol; and eugenol),are known to display modest anti-inflammatory and antinociceptiveactivity in animal and cellular models. In addition, aliphatic andalicyclic carbamates are known to be inhibitors of fatty acid amidehydrolase (FAAH), an enzyme whose inhibition is linked toanti-inflammatory effects. Thus, the individual components of theanti-inflammatory constructs of a first aspect of the invention, and thebonds that link them all together, provide a therapeutic benefit thatcan be greater than the sum of the parts.

It has now been discovered that the double and triple combinations ofthese anti-inflammatory components covalently linked together with atleast one carbamate bond yields an augmented anti-inflammatory moleculewhose net activity exceeds that of its individual building blocks. Someof these assemblies exceed the anti-inflammatory effects of thetraditional NSAIDs.

The specific structural assemblies claimed herein include:

terpene-vanilloid  Formula 1

vanilloid-polyamine-vanilloid  Formula 2

vanilloid-amino acid-terpene  Formula 3

terpene-polyamine-terpene  Formula 4

vanilloid-amino acid-vanilloid  Formula 5

terpene-amino acid-terpene  Formula 6

terpene-amino acid-vanilloid  Formula 7

In one embodiment, the carbamate-linked structures have the followinggeneral structures:

terpene-(carbamate)-vanilloid  Formula 1A

vanilloid-(carbamate)-polyamine-(carbamate)-vanilloid  Formula 2A

vanilloid-(carbamate)-amino acid-(ester)-terpene  Formula 3A

terpene-(carbamate)-polyamine-(carbamate)-terpene  Formula 4A

vanilloid-(carbamate)-amino acid-(amide)-vanilloid  Formula 5A

terpene-(carbamate)-amino acid-(ester)-terpene  Formula 6A

terpene-(carbamate)-amino acid-(amide)-vanilloid  Formula 7A

Specific examples of the components usable in construction of Formulae 1to 7 and 1A to 7A anti-inflammatory conjugates include the following.

For terpenes: The terpene of the synergistic anti-inflammatory drugconjugate is selected from the group consisting of thymol, carvacrol,menthol, geraniol, nerol, farnesol, myrtenol, cumyl alcohol,citronellol, borneol, linalool, alpha-terpineol, and perillyl alcohol.If the drug construct contains more than one terpene molecule, they maybe different or the same.

For vanilloids: The vanilloid moiety of the synergisticanti-inflammatory drug conjugate is selected from the group consistingof 4-hydroxy-3-methoxybenzyl amine commonly called vanillylamine,4-hydroxy-3-methoxybenzyl alcohol commonly called vanillyl alcohol,zingerone, [6]-paradol, and eugenol. If the drug construct contains morethan one vanilloid molecule, they may be different or the same.

For polyamines: The polyamine anti-inflammatory component is selectedfrom the group consisting of spermidine, spermine and putrescine.

For amino acids: The amino acid anti-inflammatory moiety is selectedfrom valine, leucine, isoleucine, glycine, cysteine, phenylalanine,norvaline, and other suitable amino acids known to possessanti-inflammatory activity. The amino acids can be chiral or racemic.The chirality of the chiral amino acids can be L- or R-depending on thedesired activity and release profile.

Aspect II

A second aspect of the present invention is directed to the surprisingdiscovery that conjugation of certain anti-inflammatory moieties,especially NSAIDs, vanilloids, and ketone bodies, with selected aminoacids, and optionally further conjugated with a choline bioisostere,synergistically increases the anti-inflammatory activity of theconjugate, when compared to the anti-inflammatory drug itself.

Thus, one embodiment of the present invention is directed to asynergistic anti-inflammatory drug-amino acid conjugate, comprising (a)at least one anti-inflammatory compound, and (b) at least one amino acidcovalently linked to the anti-inflammatory compound, where theanti-inflammatory activity of the conjugate is greater than the activityof the anti-inflammatory compound alone. The synergisticanti-inflammatory drug-amino acid conjugate can further incorporate acholine bioisostere (e.g., the 3,3-dimethylbutyl moiety,—OCH₂CH₂C(CH₃)₃, or it's silicon analog, —OCH₂CH₂Si(CH₃)₃), preferablyas the ester, so that another embodiment of the present invention isdirected to a synergistic anti-inflammatory drug-amino acid-cholinebioisostere conjugate, comprising (a) the anti-inflammatory drug-aminoacid conjugate above, and (b) a choline bioisosteric ester, covalentlylinked to the amino acid carboxyl of said anti-inflammatory drug-aminoacid conjugate.

In one embodiment the amino acid is covalently linked to the platformtherapeutic agent through an amino or carboxyl group as either an amideor an ester moiety.

In one embodiment the amino acid of the synergistic anti-inflammatorydrug-amino acid conjugate is selected from the group consisting ofvaline, nor-valine, leucine, iso-leucine, glycine, cysteine, proline andphenylalanine.

In one embodiment the anti-inflammatory compound is selected from thegroup consisting of non-steroidal anti-inflammatory drugs (NSAIDs),vanilloids, and ketone bodies. In a particular embodiment, the NSAID isselected from the group consisting of diclofenac, ibuprofen, naproxen,and indomethacin. The vanilloid is selected from vanillyl alcohol,phenolic hydroxyl-protected vanillyl alcohol(3-methoxy-4-acetyloxybenzyl alcohol), and vanillylamine. The ketonebody is selected from 3-hydroxybutyrate or a homologue thereof. Vanillylalcohol and vanillylamine are both known to possess anti-inflammatoryproperties. So-called “ketone bodies” of which 3-hydroxybutyric acid isa prime example, have been increasingly recognized as possessinganti-inflammatory properties.

In one embodiment, the synergistic anti-inflammatory drug-amino acidconjugate has the structure of Formula (I):

AI—NH—CHR—C(═O)O-Q¹  Formula (I)

where AI represents an anti-inflammatory drug moiety such as anNSAID-CO—, a vanillyl moiety, or 3-hydroxybutyryl, where R is selectedfrom the group consisting of hydrogen, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted aryl, andoptionally substituted heteroaryl, and where Q¹ can be selected fromhydrogen, alkyl or heteroalkyl. In one specific embodiment,Q¹=-CH₂CH₂C(CH₃)₃. Examples of this embodiment include NDH 4476, 4535,4537, 4572, 4576, 4577, 4578, 4591, 4595, 4596, 4613, 4614, 4615, 4617,4618, 4619, 4627, 4628, 4651, 4652, 4653, and 4654 as referenced herein.

In another embodiment, the synergistic anti-inflammatory drug-amino acidconjugate has the structure of Formula (II):

AI—NH—CHR—C(═O)—NH-Q²  Formula (II)

where AI represents an anti-inflammatory moiety (viz, NSAID-CO—,vanillyl alcohol-CO—, and such ketone bodies as 3-hydroxybutyryl); R isselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl; Q² is selected from hydrogen orthe vanillyl moiety (i.e., 3-methoxy-4-hydroxybenzyl), —CH₂CH₂C(CH₃)₃ or—CH₂CH₂Si(CH₃)₃. If vanillylamine (i.e., 3-methoxy-4-hydroxybenzyl-NH—)is attached to any of these anti-inflammatory amino acid platforms itconstitutes a shelf-stable, slowly metabolized moiety. However, ifvanillyl alcohol (i.e., 3-methoxy-4-hydroxybenzyl-O—) is attached, theresulting candidate pharmaceuticals are unstable unless thefree-phenolic hydroxyl is protected by acylation. Acetate is a preferredprotecting group and the derived products are suitable therapeuticcandidates. Examples of this embodiment include NDH 4479, 4483, and 4571as referenced herein.

DETAILED DESCRIPTION OF THE INVENTION Aspect I

Surprisingly, it has now been discovered that weak anti-inflammatorymoieties can be covalently linked by carbamate bonds to yield conjugateconstructs of enhanced potency for suppression of inflammation.

One aspect of the present invention is directed to an anti-inflammatoryconjugate where the anti-inflammatory component comprises at least onecompound selected from the group consisting of anti-inflammatoryterpenes, anti-inflammatory vanilloids, anti-inflammatory polyamines andanti-inflammatory amino acids.

A related aspect of the invention is directed to a method of improvingthe potency of an anti-inflammatory compound by linking it to anotheranti-inflammatory compound via a carbamate linkage, where the potency ofthe conjugate is greater than the sum of its parts.

In one embodiment of the present invention the terpene, amino acid,vanilloid, or polyamine is not employed as a single component but as anaugmenting component, covalently linked by a carbamate moiety to anotheranti-inflammatory moiety or to two other anti-inflammatory moieties,wherein they together serve to enhance or synergize performance. Theconjugates may be bifunctional (meaning just two moieties) ortri-functional (meaning three components), or higher. In addition thecarbamate linking bond itself can also convey anti-inflammatory activityto the conjugate.

Carbamate compounds are known to achieve anti-inflammation effect invivo by inhibition of fatty acid amide hydrolase. In an inhibitoryscreen against fatty acid amide hydrolase (FAAH), the inventivecarbamates were found to possess IC₅₀ values which ranged from 9 μM to 1mM for inhibition of FAAH. Some molecules were too lipophilic todissolve in the enzyme assay medium and hence could not be tested. Whilethere was no direct linear correlation between the compound's efficacyas an FAAH inhibitor and its potency in suppressing inflammation, manyof the best inflammation suppressants were also FAAH inhibitors. TheFAAH IC₅₀ values are noted with the compound examples.

Hydrolysis of the conjugates can release the terpene and any otherco-anti-inflammatories to affect the therapeutic benefit in vivo.Unfortunately, in several cases hydrolysis was too fast (of the order ofminutes) to make the compounds practical as pharmaceuticals andstabilization of the conjugate had to be addressed.

For example, as exemplified by the structures NDH4481, 4483, and 4485,if one attempts the incorporation into a conjugate of the vanilloidvanillyl alcohol (also known as 4-hydroxy-3-methoxybenzyl alcohol)through its benzyl alcohol component (the —CH₂OH), a conjugate isproduced that is rapidly hydrolyzed. It is known that 4-hydroxy benzyl-Xsystems [e.g., p-HO—Ar—CH₂—X], wherein X is a good leaving group, canrapidly decompose via a quinone methide intermediate. Capping thephenolic hydroxyl with an acetate group solves the problem, andhydrolysis lifetimes of >2 hours are then observed. This problem is notobserved with the vanillylamines when linked through their aminonitrogens; these are stable materials.

A second case of decomposition that is too rapid can be seen in NDH4590and 4593. Even though these compounds have impressive anti-inflammatoryeffects in the Mouse Ear Vesicant Model (MEVM) assay, their half-livesin sera or in any polar aqueous medium are comparatively short (hours).We have discovered that this is because the nucleophilic internalsecondary amine NH executes an intramolecular nucleophilic attack on thecarbonyl of the carbamate thereby freeing the terpene or the vanilloidcomponent. This is a controllable, or tunable, chemically-inducedhydrolysis that does not require an enzyme.

These compounds possess a terpene or vanilloid carbamate at both ends ofthe molecule in each case. With the unsymmetrical polyamine we havefound that the cyclization occurs to form the six-membered ring only(versus a seven-membered ring).

Either making a salt (such as the trifluoroacetate, hydrochloride,mesylate, or other pharmaceutically acceptable salt) or a labile amide(for example, the trifluoroacetamide, trinitrobenzamide, ortris-trifluorobenzamide) on the internal NH solves the problem, andsufficiently long hydrolysis half-lives are then observed (days). Theanti-inflammatory activity was unaffected by these stabilizingmodifications, only the time of on-set of the effect was varied (cf.NDH4616, 4622, 4630, 4631, 4635, 4637 and 4649). Half-life for releasecan be controlled or tuned as noted above, by protonation or amideformation, but it can also be controlled by varying the nature of theanti-inflammatory leaving group. For example, zingerone is released muchfaster (half-life about 2 hours) than are carvacrol or thymol(half-lives about 2 days), which in turn are released much faster thanan aliphatic terpene such as geraniol or borneol (marginal release afterseveral days). The kinetics of release follow the typical organic moiety“leaving group” abilities.

Aspect II

Surprisingly, it has now been discovered that selected amino acids (forexample valine, leucine, isoleucine, glycine, cysteine, phenylalanine,proline and norvaline) potentiate or synergize the activity ofanti-inflammatory drugs when covalently attached to the parent drugmolecules. When attached to known anti-inflammatory moieties, theseamino acids augment, or synergize, the anti-inflammatory potency,provide a bio-compatible controlled-release, and permit adjustment ofthe pharmacologic properties of the parent anti-inflammatory drug.

Thus, in a second aspect of the invention, the amino acid can be used asa “capping” group on an anti-inflammatory such as a NSAID, a vanillylalcohol or a vanillylamine. In one embodiment, the amino acid can beattached through its amino group to a carboxyl group in the platformanti-inflammatory molecule leaving a pendant carboxyl from the aminoacid which can be free (Q¹=H) or can be esterified (Q¹=alkyl) forenhancement of properties or for ease of handling. A preferred alkylgroup is a choline mimic, such as —CH₂CH₂C(CH₃)₃ or its siliconbioisostere, —CH₂CH₂Si(CH₃)₃. In one specific embodiment, constructs orscaffolds of this type can be characterized as shown in Formula (I):

AI—NH—CHR—C(═O)O-Q¹  Formula (I)

In a second embodiment, herein called Formula (II), when oneanti-inflammatory compound contains an amino group, such as in thetransient receptor potential cation channel subfamily V member 1 (TRPV1)inhibitor vanillylamine, the amino acid augmentation moiety can belinked via its carboxyl resulting in a pendant amino to which can beattached a second anti-inflammatory component such as an NSAID-CO—, avanillyl alcohol-CO—, or a 3-hydroxybutyryl (3-HB) unit (asrepresentative of a ketone body).

AI—NH—CHR—C(═O)—NH-Q²  Formula (II)

NDH 4571 in which 3-HB is mounted on a valine platform linked to avanilloid, displayed a 69% suppression of chloroethyl ethyl sulfide(CEES)-induced inflammation at the standard test dosage in the MEVM,considerably higher than any of the fragment pieces of that conjugate.

Present Embodiments

One aspect of the invention is directed to an anti-inflammatorydrug-amino acid conjugate, comprising: (a) at least oneanti-inflammatory compound conjugated with (b) an augmenting moietycomprising an anti-inflammatory amino acid selected from the groupconsisting of valine, nor-valine, leucine, iso-leucine, glycine,cysteine, proline and phenylalanine; wherein conjugation is via thenitrogen atom of the amino acid of the augmenting moiety; and whereinthe anti-inflammatory activity of the conjugate is greater than the sumof its parts. Preferably the anti-inflammatory drug-amino acid conjugatecontains a core anti-inflammatory amino acid, with supplementalanti-inflammatory agents or moieties covalently attached to both thecarboxylic acid group and the amino group, where these supplementalanti-inflammatory morieties or agents are not amino acids. The inventiveconjugate can also have a dipeptide core rather than a monomeric aminoacid, but the core is not a higher oligopeptide or a protein. Thus, notonly the anti-inflammatory monomeric amino acids but also di-peptidescontaining at least one of the anti-inflammatory amino acids constituteuseful bi-functional platforms to carry the supplementalanti-inflammatory moieties. Such dipeptides include, for example, valylvaline, valyl glycine, valyl alanine, valyl proline, valylphenylalanine, glycyl valine, prolyl valine, phenylalanyl valine,isoleucyl valine, alanyl valine, glycyl proline, prolyl glycine, glycylphenylalanine, phenylalanyl glycine, prolyl phenylalanine, phenylalanylproline and related bis-amino acid units.

With regard to amino acid chemistry it is commonly understood that theverb “to conjugate” refers to reacting an amino substituent in oneconjugation partner with a carboxylic acid substituent (or suitablyactivated carboxylate group) on a second conjugation partner, withelimination of a small molecule (typically water), thereby joining thetwo partners via an amide bond. Similarly, the verb “to conjugate” alsosignifies “to join together” in grammar, as in “to conjugate a verb”.Thus in chemistry, a conjugate is a chemical compound that has beenformed by the joining of two or more compounds.

In one embodiment the amino acid of the anti-inflammatory drug-aminoacid conjugate is selected from the group consisting of valine, glycine,proline and phenylalanine. Preferably the amino acid is valine orproline or phenylalanine. More preferably the amino acid is valine orphenylalanine. In one embodiment the amino acid is valine. In anotherembodiment the amino acid is phenylalanine.

In one embodiment the augmenting moiety is an amino acid ester of thecholine bioisosteres HOCH₂CH₂C(CH₃)₃ or HOCH₂CH₂Si(CH₃)₃, or an aminoacid amide of the choline bioisosteres H₂NCH₂CH₂C(CH₃)₃ orH₂NCH₂CH₂Si(CH₃)₃. In another embodiment the augmenting moiety is avaline ester or amide. In another embodiment the augmenting moiety is aphenylalanine ester or amide. In another embodiment the augmentingmoiety is a proline ester or amide. In one embodiment the augmentingmoiety is an amino acid ester or amide of a vanilloid. Preferably thevanilloid is selected from the group consisting of vanillyl alcohol,vanillyl amine and phenol-protected derivatives thereof.Phenol-protected derivatives include O-acylated analogs, such asacetyloxy (also known as “acetoxy”) and benzoyloxy compounds.

In one embodiment the anti-inflammatory compound is selected from thegroup consisting of non-steroidal anti-inflammatory drugs (NSAIDs),anti-inflammatory vanilloids and ketone bodies. In one embodiment theNSAID is selected from the group consisting of diclofenac, ibuprofen,naproxen, and indomethacin. In another embodiment the vanilloid isselected from the group consisting of vanillyl alcohol,3-methoxy-4-acetyloxybenzyl alcohol, and vanillylamine. In yet anotherembodiment the ketone body is selected from the group consisting of3-hydroxybutyrate and homologues thereof. “Ketone bodies” such as3-hydroxybutyrate and acetoacetate are produced as metabolites of fattyacids in the liver. 3-Hydroxybutyrate has inherent anti-inflammatoryactivity. For purposes of the present disclosure, a “homologue” isdefined as a compound belonging to a series of compounds differing fromeach other by one or more methylene (—CH₂—) groups, for example by asingle methylene group. Thus 4-hydroxypentanoate and 3-hydroxypentanoateare both higher homologues of 3-hydroxybutyrate, depending on where inthe carbon chain the methylene group has been inserted with respect tothe hydroxy-bearing carbon of 3-hydroxybutyrate.

In addition to NSAIDs, vanilloids and ketone bodies, other usefulanti-inflammatory compounds include anti-inflammatory terpenes (e.g.,geraniol, thymol, carvacrol, etc), anti-inflammatory hydroxy-cinnamicacids (e.g., ferulic acid, caffeic acid, and p-coumaric acid),anti-oxidants (e.g., cathecins/catechins and flavanols),indole-3-carbinol, pentoxifylline, and anti-inflammatory fatty acids(e.g., ricinoleic, palmitoleic, and docosahexaenoic).

A related aspect of the invention is directed to an anti-inflammatorydrug-amino acid conjugate comprising: (a) an anti-inflammatory compoundconjugated with (b) an augmenting moiety comprising an amino acid esteror amide, wherein conjugation is via the nitrogen atom of the amino acidof the augmenting moiety; wherein the amino acid ester or amide isselected from the group consisting of esters and amides of valine,glycine, proline and phenylalanine, wherein the anti-inflammatorycompound is selected from the group consisting of (1) the non-steroidalanti-inflammatory drugs diclofenac, ibuprofen, naproxen, andindomethacin; (2) the vanilliods vanillyl alcohol,3-methoxy-4-acetyloxybenzyl alcohol, and vanillylamine; and (3) theketone bodies 3-hydroxybutyrate and homologues thereof; and wherein theanti-inflammatory activity of the conjugate is greater than the sum ofits parts.

One aspect of the invention is directed to an anti-inflammatorydrug-amino acid conjugate having the structure of Formula (I),AI—NH—CHR—C(═O)—O-Q¹, wherein AI represents an anti-inflammatory drugmoiety selected from the group consisting of an NSAID-CO— moiety, avanillyl-CO— moiety and a 3-hydroxybutyroyl moiety; wherein R isselected from the group consisting of hydrogen, isopropyl and benzyl;and wherein Q¹ is selected from the group consisting of alkyl andheteroalkyl. In this aspect the augmenting moiety is ananti-inflammatory amino acid ester. In one embodiment Q¹ is—CH₂CH₂C(CH₃)₃ or —CH₂CH₂Si(CH₃)₃. In one embodiment, for the NSAID-CO—moiety, the NSAID is selected from the group consisting of diclofenac,naproxen and indomethacin.

A related aspect of the invention is direct to an anti-inflammatorydrug-amino acid conjugate having the structure of Formula (II),AI—NH—CHR—C(═O)—NH-Q², wherein AI represents an anti-inflammatory drugmoiety selected from the group consisting of an NSAID-CO— moiety, avanillyl-CO— moiety and a 3-hydroxybutyroyl moiety; wherein R isselected from the group consisting of hydrogen, isopropyl and benzyl;and Q² is 3-methoxy-4-hydroxybenzyl, —CH₂CH₂C(CH₃)₃, or —CH₂CH₂Si(CH₃)₃.In this aspect the augmenting moiety is an anti-inflammatory amino acidamide. Preferably the anti-inflammatory amino acid amide is not anoligopeptide or a protein, but is a single anti-inflammatory amino acid,or at most a dipeptide containing at least one anti-inflammatory aminoacid, reacted with an organic amine with the elimination of water toform an amide bond. The organic amine is preferably a primary orsecondary amine. In one embodiment, for the NSAID-CO— moiety, the NSAIDis selected from the group consisting of diclofenac, ibuprofen, naproxenand indomethacin.

Another aspect of the invention is directed to a method of increasingthe activity of an anti-inflammatory drug, comprising conjugating theanti-inflammatory drug with an amino acid augmenting moiety to providean amino acid conjugate of Formula (I) or Formula (II). In oneembodiment Q¹ of Formula (I) is selected from the group consisting of—CH₂CH₂C(CH₃)₃ and —CH₂CH₂Si(CH₃)₃. In one embodiment Q² of Formula (II)is —CH₂CH₂C(CH₃)₃ or —CH₂CH₂Si(CH₃)₃.

Another aspect of the invention is directed to an anti-inflammatorydrug-amino acid conjugate selected from the group consisting of:

Compounds such as NDH4481 and NDH4535 are simple ethyl esters ratherthan the more complex 3,3-dimethylbutyl or vanillyl alcohol esters. Itis now recognized that ethanol itself possesses anti-inflammatoryactivity in humans, and therefore serves as an anti-inflammatoryaugmenting moiety in the anti-inflammatory drug-amino acid conjugate.

Preferably the anti-inflammatory drug-amino acid conjugate is selectedfrom the group consisting of:

In one embodiment the anti-inflammatory drug-amino acid conjugate isNDH4479. In another embodiment the anti-inflammatory drug-amino acidconjugate is NDH4481. In yet another embodiment the anti-inflammatorydrug-amino acid conjugate is NDH4483. In a further embodiment theanti-inflammatory drug-amino acid conjugate is NDH4486.

EXAMPLES Materials and Methods

All reactants and solvents used were of the highest purity commercialgrade and were employed without further purification. All amino acidsused herein were the L-amino acids and were purchased from Sigma-Aldrich(Saint Louis, Mo.). The 2-(2-methoxynaphthalene-6-yl) propanoic acid(naproxen) used was the (S)-enantiomer. All other reagents were used asracemates, unless otherwise noted. All reactions were performed inoven-dried apparatus under a nitrogen atmosphere, unless otherwisenoted. All solvents used were anhydrous, unless otherwise noted. NMRspectra were recorded on a Bruker multinuclear spectrometer and chemicalshifts are reported as ppm using tetramethylsilane (TMS) as an internalstandard. ¹H NMR spectra were recorded at 500 MHz, while ¹³C NMR spectrawere recorded at 125 MHz. Elemental analyses were performed at Intertek(Whitehouse, N.J.). All thin layer chromatography (TLC) was performed onAnaltech silica gel plates (250 microns).

Biological Evaluations Ellman Assay

The modified Ellman assay for inhibition of acetylcholinesterase (AChE)and the mouse ear vesication assay (MEVA) have been described in detailby us (see S. C. Young et al, J Appl Tox, 2012, 32: 135-141). AChE (TypeV-S from electrophorus electricus), acetylthiocholine iodide (ATChI),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and tacrine from EMDChemicals (Gibbstown, N.J.). Cholinesterase inhibition was assayedspectrophotometrically at 412 nm according to Ellman's method. Assayswere performed in polystyrene 96-well plates (Corning 96-well flattransparent) and a conventional micro-plate reader was employed forkinetic readings (Tecan Infinite 200 multimode). The following reagentswere added to the wells: 200 μL of 0.5 mM DTNB in sodium phosphatebuffer (100 mM, pH 8), 30 μL of inhibitor stock solution in methanol, 20μL of 1.25 units/mL of AChE in sodium phosphate buffer (20 mM, pH 7),and 50 μL of 3 mM ATCh in buffer (100 mM, pH 8). Immediately after thesubstrate was added, the absorption signal was measured at 30 sintervals over 5 min at 25° C. Percentage inhibition was calculatedrelative to a negative control (methanol). The background signal wasmeasured in control wells containing every reagent except for thesubstrate. IC₅₀ values were obtained from a minimum of eightconcentrations in duplicate and by fitting the experimental data with adose-response curve using Prism software (Version 5.00, GraphPadSoftware, San Diego, Calif.).

Mouse Ear Vesicant Model (MEVM)

Animal studies were approved by the Rutgers University InstitutionalAnimal Care and Use Committee and received human care in compliance withthe institution's guidelines, as outlined in the Guide for the Care andUse of Laboratory Animals of the National Academy of Sciences. Compoundswere assessed as inhibitors of inflammation using the MEVM as previouslydescribed (Casillas, R P., et al., Therapeutic approaches todermatotoxicity by sulfur mustard. 1. Modulaton of sulfurmustard-induced cutaneous injury in the mouse ear vesicant model, J.Appl. Toxicol., 2000, 20, Suppl 1, S145-51), except that female CD-1mice (4-6 weeks old) were used. Either CEES, chloroethyl ethyl sulfide(65 μmoles) or TPA, 12-O-tetradecanoylphorbol-13-acetate, (1.5 nmol) wasused to induce inflammation. To evaluate each compound, ears (3-4 miceper group) were treated with 20 μL, of vehicle control (methylenechloride or acetone) or the test compound (1.5 μmol) in 20 μL, of theappropriate vehicle. After 5 h, mice were euthanized and ear punches (6mm in diameter) were taken and weighed. Once the raw data were obtained,masses of ear punches were averaged and the percent reduction ofvesicant-induced edema and inflammation was calculated using the methodof Casillas et al. Raw data were analyzed using a one-way ANOVA toevaluate statistical significance (P<0.05).

Inflammation suppression, if observed, is of course dose related but isreported herein only at the standard dose mentioned above. On occasion,mostly with ibuprofen analogs, the vesicant-induced damage is augmentedby the candidate anti-inflammatory and these substances are designatedas irritants. Also, in some cases the anti-inflammatory candidatesuppresses the mean weight of the ear punches from the test ears belowthat observed with the untreated control and these results are statedas >100% suppression.

Examples of Aspect I

The bifunctional and tri-functional conjugates of Aspect I of theinvention were prepared and tested in a standard in vivo MEVM assay fortheir efficacy compared to that of the parent terpene, amino acid,polyamine, or vanilloid from which each was assembled. Terpeneinflammation suppression scores (average of TPA-induced and CEES-inducedinjuries) ranged from myrtenol (6%), thymol (14%), carvacrol (15%),cumyl alcohol (16%), geraniol (35%), menthol (38%), perillyl alcohol(43%), and farnesol (69%). All terpenes, except farnesol, hadinflammation suppression scores less than 45%. The inflammation scoresof typical vanilloids were similarly low and none exceeded 40%, e.g.,vanillin (11%), vanillyl alcohol (31%), and vanillylamine (35%). In thisassay inflammation suppression scores for the amino acids and thepolyamines were under 30%. The traditional NSAIDs were under 40% ininflammation suppression scores, e.g., ibuprofen (−23%, anirritant/inflammation inducer), S-naproxen (31%), piroxicam (32%),diclofenac (37%), and indomethacin (39%).

The synergistic effects of combination of weakly potentanti-inflammatory components into conjugates are readily evident in thecompounds of the invention. As an example of the Formula 1 class recitedearlier herein, the terpene carvacrol by itself displayed inflammationsuppression of 19% and 10% for CEES and TPA-induced inflammationrespectively while its carbamate conjugate with vanillylamine (NDH4574)showed a significantly improved suppression of 89% and 88% (CEES andTPA).

Another Formula 1 conjugate combines the terpene linalool to thevanilloid, vanillylamine, to yield the construct (NDH4624) whichdisplayed a 92% suppression of CEES-induced inflammation.

As another example of the Formula 1 conjugate, the terpene (geraniol)coupled to the vanilloid (vanillylamine) by a carbamate linkage anddesignated as NDH4484 had a 64% suppression (CEES-induced injury) and a71 μM inhibition of fatty acid amide hydrolase (FAAH).

Similarly, a Formula 1 example involving perillyl alcohol showed thesame trend with an inflammation suppression score of 43% (for the parent“free” terpene) while its carbamate conjugate with vanillylamine(NDH4498) showed an enhanced suppression of 53% (CEES) and 76% (TPA).This carbamate showed an IC₅₀ for inhibition of fatty acid amidehydrolase (FAAH) of 14 μM.

The Formula 2 conjugates (vanilloid-polyamine-vanilloid) can beillustrated by the construct of eugenol-spermidine-eugenol (NDH4635)which displays an inflammation suppression of 73% (CEES-inducedinflammation) and zingerone-spermidine-zingerone

(NDH4637) which displays an 89% suppression against CEES-induced and 93%suppression against TPA-induced inflammation. The salt is needed to slowhydrolytic release of the zingerone.

A tri-functional conjugate, NDH4486, (a Formula 3 example), in which theterpene geraniol (35% inflammation suppression score as unconjugatedterpene molecule) was linked to the amino acid valine by a carbamatelinkage and thence to the vanilloid vanillylamine, proved especiallypotent (91%) in suppression of TPA-induced inflammation in the mouseear.

As an example of the Formula 4 conjugates, when carvacrol was linked asa bis-derivative to the well-known polyamine, spermidine, theinflammation suppression of the combined moiety increased to 71% againstCEES-induced and 110% against TPA induced inflammatory injury (seeNDH4593 shown below). The naturally occurring polyamines such asputrescine, spermidine, and spermine can display anti-inflammatoryeffects either as free molecular entities or as conjugates with alltrans-retinoic acid. These effects are clearly augmented by attachmentto terpenes through carbamate linkages.

In addition, in a Formula 4 example, thymol displayed an inflammationsuppression score of 14% while its carbamate conjugate with spermidine(NDH4590) showed an impressive and complete inflammation suppression of100% against either CEES or TPA-induced injury.

Slower to hydrolyze and to liberate the terpene moiety are thetrifluoroacetate salts or amides as exemplified by thecarvacrol-spermidine conjugate, NDH4622, with

83% (CEES) and 100% (TPA). The similarly stabilized carvacrol-sperminebis trifluoroacetate salt conjugate, NDH4631, was assayed with 84%(CEES) and 89% (TPA) values.

The covalently-attached trifluoroacetyl (as an amide) yields a verystable thymol-spermidine conjugate, NDH4616, which retained considerableanti-inflammatory activity, 76% (TPA).

As an example of a Formula 5 compound, NDH4483 links two vanilloid units(vanillyl alcohol and vanillylamine) to a core valine unit. Theinflammation suppression was 67% (TPA) and the FAAH IC₅₀ was 1.0 mM. Thehydrolysis half-life without the acetyl group attached to thepara-hydroxyl of the vanillyl alcohol moiety was under 5 minutes inphysiological saline.

A modification of this Formula 5 compound in which the vanillylamineportion has been deleted (NDH4481) had the same hydrolyticinstability-unless the p-hydroxyl group was acetylated—

and possessed the same FAAH IC₅₀ of 1.0 mM but with a slightly improvedinflammation suppression of 72% (CEES-induced) and 93% (TPA-induced).

As an example of a Formula 6 compound, NDH 4648 joins the terpenecarvacrol to the amino acid valine by a carbamate bond and thence joinsthe terpene farnesol to that same amino acid by an ester bond.

As an example of a Formula 7 compound, NDH 4486 links the terpenegeraniol to the amino acid valine by a carbamate bond and thence joinsthe vanilloid vanillylamine to that same amino acid by an amide bond.The resulting conjugate showed an inflammation suppression of 91%(TPA-induced).

Aspect I Synthesis

The compounds of the invention were synthesized by the pathways outlinedin Schemes 1, 2, 3, 4, and 5, using the application of a thiazolide totransfer the —COOR unit to the polyamine, amine, or amino acid unit. Theactivated thiazoline is synthesized as shown in Scheme 2 if the terpenebeing transferred has a secondary hydroxyl group, otherwise the pathwayas shown in Scheme 1 is suitable. Scheme 3 shows the transfer pathwayfor —COOR moiety to the polyamines; similar chemistry applies fortransfer to amino acids. Scheme 3 shows how the internal secondary NH inthe polyamine can have its nucleophilicity suppressed by salt formationor acetamide formation in order to prevent auto-decomposition. Scheme 4shows how terpene and/or vanilloid moieties are transferred to an aminoacid platform compound. Scheme 5 shows how terpene moieties are directlylinked to vanilloid moieties (vanillylamine as example) to generateconjugates of Formula 1.

Specific examples selected from the seven Formulae of conjugates havebeen presented herein but these do not represent the limits of thestructural possibilities. Table 1 provides examples of a wider range ofsynthetic targets obtainable by the experimental methods describedherein and consistent with the seven Formulae of conjugates disclosedherein. Systematic names are provided for these anti-inflammatories.Table 1 includes the compounds discussed herein.

TABLE 1 Structural diversity consistent with the formulae of Aspect Iconjugates of the invention NDH4616:

5-methyl-2-(propan-2-yl)phenyl[3-(trifluoroacetyl{4-[(5-methyl-2-(propan-2-yl)phenoxycarbonyl)amino]butyl}amino)propyl]carbamate NDH4622:

2-methyl-5-(propan-2-yl)phenyl [3-({4-[(2-methyl-5-(propan-2-yl)phenoxycarbonyl)amino]butyl}amino)propyl]carbamate trifluoroaceticacid salt NDH4630:

1,7,7-trimethylbicyclo[2.2.1]hept-2-yl [3-({4-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yloxycarbonyl)amino]butyl}amino)propyl]carbamate trifluoroacetic acid saltNDH4635:

2-methoxy-4-(prop-2-en-1-yl)phenyl [3-({4-[(2-methoxy-4-(prop-2-en-1-yl)phenoxycarbonyl)amino]butyl}amino)propyl]carbamate trifluoroaceticacid salt NDH4637:

2-methoxy-4-(3-oxobutyl)phenyl [3-({4-[(2-methoxy-4-(3-oxobutyl)phenoxycarbonyl)amino]butyl}amino)propyl]carbamatetrifluoroacetic acid salt NDH4649:

5-methyl-2-(propan-2-yl)phenyl[3-({4-[(5-methyl-2-(propan-2-yl)phenoxycarbonyl)amino]butyl}amino)propyl]carbamate trifluoroaceticacid salt NDH4631:

bis(5-isopropyl-2-methylphenyl)((butane-1,4-diylbis(azanediyl))bis(propane- 3,1-diyl))dicarbamateNDH4638:

(S)-(1R,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl2-(((2-methoxy-4-(3- oxobutyl)phenoxy)carbonyl)amino)-3-methylbutanoateNDH4639:

(S)-(1R,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl2-(((2-methoxy-4-(3- oxodecyl)phenoxy)carbonyl)amino)-3-methylbutanoateNDH4640:

(S)-(1R,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(((4-allyl-2-methoxyphenoxy)carbonyl)amino)-3-methylbutanoate NDH4641:

(S)-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl 2-(((4-allyl-2-methoxyphenoxy)carbonyl)amino)-3-methylbutanoate NDH4642:

(S)-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl2-(((2-methoxy-4-(3- oxobutyl)phenoxy)carbonyl)amino)-3-methylbutanoateNDH4647:

(S)-(1R,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl2-(((4-isopropyl-2- methylphenoxy)carbonyl)amino)-3-methylbutanoateNDH4648:

(S)-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl 2-(((4-isopropyl-2-methylphenoxy)carbonyl)amino)-3-methylbutanoate NDH4486:

(S,E)-3,7-dimethylocta-2,6-dien-1-yl (1-((4-hydroxy-3-methoxybenzyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

Preparation of Trifluoroacetic Acid Salts of Polyamines A) Formation ofProtected Carbamates

General Procedure (NDH4616, 4622, 4630, 4631, 4635, 4637 and 4649)

The polyamine (spermidine or spermine) was weighed into a round bottomflask containing a stirring bar. The amine was dissolved in drydichloromethane (CH₂Cl₂) (10 mL/mmol). To the stirred solution at roomtemperature were added two equivalents of an alkyl or aryl2-thioxo-1,3-thiazolidine-3-carboxylate (hereafter referred to as athiazolidine carbamate) which rendered a yellow solution. The progressof the reaction was monitored by the loss of the yellow color as well asby TLC which revealed the release of 2-mercaptothiazoline (MTA) and thedisappearance of the thiazolidine carbamate. After the first step wascomplete triethylamine (1 equivalent) was added to the reaction flaskfollowed by the addition of Boc anhydride (Boc₂) (1 equivalent). Oncethe second step was complete, as noted by TLC, the reaction solution wasdiluted with CH₂Cl₂, and the resulting solution was extracted with 1NHCl and then saturated NaCl. The organic layer was dried over MgSO₄(anhydrous), filtered, concentrated on the rotary evaporator and driedunder vacuum. The crude material was covered with a solution of 7:3,hexanes/ethyl acetate (EtOAc) in order to crystallize out the releasedMTA. The supernatant was drawn off and concentrated. The product waspurified by column chromatography on silica gel eluting with 7:3,hexanes/EtOAc.

1. NDH 4622: R_(f)=0.32 (7:3, hexanes/EtOAc); Yield=76%.

2. NDH 4630: R_(f)=0.39 (7:3, hexanes/EtOAc); Yield=57%.

3. NDH 4649: R_(f)=0.27 (7:3, hexanes/EtOAc); Yield=63%.

4. NDH 4631: Removal of MTA from the crude material was accomplishedusing 3:2, hexanes/EtOAc. Column purification was carried out using96:4, CH₂Cl₂/acetone as eluant. R_(f)=0.25 (96:4, CH₂Cl₂/acetone);Yield=83%.

5. NDH 4635: The crude material was purified by column chromatography,without removing MTA, first using 98:2, CH₂Cl₂/MeOH and for the secondcolumn 96:4, CH₂Cl₂/acetone. R_(f)=0.21 (96:4, CH₂Cl₂/acetone):Yield=77%.

6. NDH 4637: The crude material was purified by column chromatography,without removing MTA, using a gradient of 94:6, CH₂Cl₂/acetone to 9:1,CH₂Cl₂/acetone and then 97:3, CH₂Cl₂/MeOH. R_(f)=0.06 (95:5,CH₂Cl₂/acetone); Yield=100%.

7. NDH 4616: Upon completion of the first step, 1.5 equivalents of ethyltrifluoroacetate were added in place of the Boc₂ and triethylamine, andthe reaction mixture was stirred overnight. The product crystallized outof the reaction, and was collected by suction filtration and rinsed withCH₂Cl₂. Exact mass (ESI) calculated for C₂₉H₄₄N₃O₄ [M+H]498.3326 found498.3334. The exact mass represents the compound resulting from loss ofthe trifluoroacetyl group. R_(f)=0.70 (9:1, CH₂Cl₂/MeOH): mp=190-191°C.; Yield=51%.

B) Formation of Trifluoroacetic Acid (TFA) Salts

General Procedure (NDH4616, 4622, 4630, 4631, 4635, 4637 and 4649)

The Boc-containing protected carbamate was dissolved in anhydrous CH₂Cl₂(20 mL/mmol). Trifluoroacetic acid (4 mL/mmol) was added at roomtemperature. The reaction solution was stirred, and the progress of thereaction was monitored by TLC (7:3, hexanes/EtOAc). The deprotection wascomplete in 1-2 h. The volatiles were removed by distillation employingan aspirator vacuum. The residue was frozen on liquid N₂ and dried underhigh vacuum. The dry product was covered with diisopropyl ether and thesolid that separated was triturated and collected by suction filtration.

1. NDH 4622: Exact mass (ESI) calculated for C₂₉H₄₄N₃O₄ [M+H] 498.3326.found 498.3334. White powder; Yield=68%.

2. NDH 4631: Exact mass (ESI) calculated for C₃₂H₅₁N₄O₄ [M+H] 555.3905.found 555.3896. White solid; Yield=72%.

3. NDH 4649: Exact mass (ESI) calculated for C₂₉H₄₄N₃O₄ [M+H] 498.3326.found 498.3324. White solid; Yield=95%

4. NDH 4630: Exact mass (ESI) calculated for C₂₉H₅₂N₃O₄ [M+H] 506.3952.found 506.3973. Viscous oil; Yield=100%.

5. NDH 4635: The reaction was monitored by using 98:2, CH₂Cl₂/MeOH asthe TLC solvent. The crude residue was covered with diethyl ether andtriturated in order to isolate the pure product. Exact mass (ESI)calculated for C₂₉H₄₀N₃O₆ [M+H] 526.2912. found 526.2944. White powder;Yield=88%.

6. NDH 4637: The reaction was monitored using 96:4, CH₂Cl₂/acetone asthe TLC solvent. The crude residue was covered with diethyl ether andtriturated in order to isolate the pure product. Exact mass (ESI)calculated for C₃₁H₄₄N₃O₈ [M+H] 586.3123. found 586.3141. White solid;Yield=85%.

NMR Data 1) NDH 4622

¹HNMR (methanol-d₄) δ: 7.14-7.10 (m, 2H, 2×ArH-3), 7.02-6.98 (m, 2H,2×ArH-4), 6.88-6.83 (m, 2H, 2×ArH-6), 3.22 (bt, 2H, HNCH ₂CH₂CH₂N),3.11-3.02 (m, 4H, CH ₂NHCH ₂), 2.89-2.81 (m, 2H, 2×HC(CH₃)₂), 2.15-2.11(overlapping singlets, 6H, 2×Ar—CH₃), 1.97-1.89 (m, 2H, NHCH₂CH ₂CH₂NH),1.79-1.69 (m, 2H, NHCH₂CH ₂CH₂—CH₂NHCO), 1.69-1.60 (m, 2H, NHCH₂CH₂CH₂CH₂NHCO), and 1.22-1.18 (overlapping doublets, 12H, ³J=6.9 Hz,2×ArCH(CH ₃)₂). Note: The protons OCHNCH ₂CH₂CH₂NH are masked beneaththe methanol-d₄ CH₃ peak centered at δ3.30.

2) NDH 4630

¹HNMR (CDCl₃+D₂O) δ: 3.31 (bt, 2H, OCHNCH ₂CH₂CH₂NH), 3.17 (t, 2H,³J=6.70 Hz, NHCH₂CH₂CH₂CH ₂NHCO), 3.05-2.92 (m, 4H, CH₂NHCH₂), 2.36-2.24(m, 2×1H, 3-H exo), 1.98-1.90 (m, 2H, NHCH₂CH ₂CH₂NH), 1.90-1.55 (m,10H, NHCH₂CH ₂CH ₂CH₂NHCO, 2×bornyl H-4, 2×bornyl H-5 exo and 2×bornylH-6 endo), 1.30-1.16 (m, 4H, 2×bornyl H-5 endo and 2×bornyl H-6 exo),1.00-0.94 (m, 2H, 2×bornyl H-3 endo), 0.88-0.86 (bd, 6H, 2×bornyl C-7CH₃), 0.85-0.83 (bd, 6H, 2×bornyl C-7 CH₃) and 0.81 (bs, 6H, 2×bornylC-1 CH₃). Note: The bornyl C-2 protons are masked beneath the D₂O peak.

3) NDH 4631

¹HNMR (methanol-d₄) δ: 7.16-7.10 (bd, 2H, 2×ArH-3), 7.04-6.98 (m, 2H,2×ArH-4), 6.89-6.84 (bd, 2H, 2×Ar-6), 3.11-2.99, (m, 8H, CH ₂NCH₂CH₂CH₂CH ₂NCH ₂), 2.90-2.81 (m, 2H, 2×CH(CH₃)₂), 2.14 (bs, 6H,2×ArCH₃), 1.97-1.89 (m, 4H, 2×NCH₂CH ₂CH₂N), 1.80-1.72 (m, 4H, NCH₂CH₂CH ₂CH₂N), and 1.21 (bd, 12H, ³J=6.95 Hz, 2×HC(CH ₃)₂). Note: Theprotons 2×OCNHCH ₂ are masked beneath the methanol-d₄ CH₃ peak centeredat δ3.30.

4) NDH 4635

¹HNMR (methanol-d₄) δ: 6.98-6.90 (2 sets of doublets, 2H, ³J=8.0 and8.05 Hz, 2×ArH-6), 6.90-6.84 (2 sets of doublets, 2H, ⁴J=1.65 Hz,2×ArH-3), 6.79-6.71 (m, 2H, 2×ArH-5), 60.1-5.90 (m, 2H, 2×CH₂═CH),5.12-5.01 (m, 4H, 2×CH2═CH), 3.80 (s, 3H, Ar—OCH₃), 3.78 (s, 3H,Ar—OCH₃), 3.36 (overlapping doublets, 4H, ³J=6.65 Hz, 2×ArCH ₂—CH═CH₂),3.22-3.16 (m, 2H, NHCH₂CH₂CH₂CH ₂NHCO), 3.12-3.00 (m, 4H, CH ₂NHCH ₂),1.97-1.87 (m, 2H, NCH₂CH ₂CH₂N), 1.80-1.68 (m, 2H, NHCH₂CH ₂CH₂CH₂NHCO),and 1.67-1.57 (m, 2H, NHCH₂CH₂CH ₂CH₂NHCO). Note: The protons OCHNCH₂CH₂CH₂NH are masked beneath the methanol-d₄ CH₃ peak centered at δ3.30.

5) NDH 4637

¹HNMR (methanol-d₄) δ: 6.96-6.87 (m, 4H, 2×ArH-3 and 2×ArH-6), 6.80-6.73(m, 2H, 2×ArH-5), 3.84-3.74 (m, 6H, 2×Ar-OCH₃), 3.21-3.14 (m, 2H, OCHNCH₂CH₂CH₂NH), 3.12-3.00 (m, 4H, CH ₂NHCH ₂), 2.88-2.76 (m, 8H, 2×ArCH₂CH₂CO), 2.12-2.11 (overlapping singlets, 6H, 2×COCH ₃), 1.96-1.85 (m,2H, NCH₂CH ₂CH₂N), 1.79-1.68 (m, 2H, NHCH₂CH ₂CH₂CH₂NHCO) and 1.67-1.58(m, 2H, NHCH₂CH₂CH ₂CH₂NHCO). Note: The protons OCHNCH ₂CH₂CH₂NH aremasked beneath the methanol-d₄ CH₃ peak centered at δ3.30.

6) NDH 4649

¹HNMR (methanol-d₄) δ: 7.24-7.13 (m, 2H, ArH-3), 7.06-6.95 (m, 2H,ArH-4), 6.86-6.75 (m, 2H, ArH-6), 3.25-3.21 (m, 2H, NHCH₂CH₂CH₂CH₂NHCO), 3.13-2.98 (m, 6H, 2×CH(CH₃)₂ and CH ₂NHCH ₂), 2.30 (bs, 6H,2×ArCH₃), 1.99-1.88 (m, 2H, NCH₂CH ₂CH₂N), 1.82-1.70 (m, 2H, NHCH₂CH₂CH₂CH₂NCO) and 1.70-1.60 (m, 2H, NHCH₂CH₂CH ₂CH₂NCO). Note: The protonsOCNCH ₂CH₂CH₂NH are masked beneath the methanol-d₄ CH₃ peak centered atδ3.30.

7) NDH 4616

¹HNMR (acetone-d₆) δ: 7.22 (bs, 2H (partially exchanged), 2×NH), 7.17(apparent triplet, 2H, ³J=7.4 Hz, 2×ArH-3), 6.98 (apparent triplet, 2H,³J=7.8 Hz, 2×ArH-4), 6.87 (s, 1H, ArH-6), 6.84 (s, 1H, ArH-6), 3.41-3.35(m, 2H, HNCH₂CH₂CH ₂N), 3.28-3.16 (m, 6H, NHCH ₂CH₂CH₂NCH ₂CH₂CH₂CH₂NH), 3.10-3.04 (m, 2H, Ar—CH(CH₃)₂), 2.08 (m, 2H, HNCH₂CH₂CH₂N),1.93-1.84 (m, 2H, —NCH₂CH₂CH₂CH₂NH—), 1.72-1.65 (m, 2H, —NCH₂CH₂CH₂CH₂NH—) and 1.20-1.12 (overlapping doublets, 12H, ³J=6.85 Hz, 2×ArCH(CH₃)₂).

Preparation of Valine-Based Compounds A) Carbamate Formation

A flask containing a stirring bar was charged with the N-acylthiazolidine-2-thione (1 eq) and L-valine (1.05 eq). To the flask wasadded THF (5 mL/mmol of the N-acyl thiazolidine-2-thione), and themixture was stirred until all the N-acyl thiazolidine-2-thionedissolved. Water (5 mL/mmol) was then added followed byN,N-diisopropylethylamine (2 eq), and the resulting two-phase system wasstirred vigorously at room temperature.

The progress of the reaction was monitored by TLC (9:1, CH₂Cl₂/MeOH,v/v) and by the disappearance of the yellow color originating from theN-acyl thiazolidine-2-thione. When the reaction was complete, thesolution was diluted with CH₂Cl₂ and extracted with 1N HCl. The organiclayer was concentrated on the rotary evaporator, the residue taken up inEt₂O, and the resulting ether layer was extracted with saturated NaHCO₃.The aqueous layer was then washed with Et₂O. The aqueous phase wasacidified to pH=2-3 with 4N HCl. The resulting mixture was extractedwith CH₂Cl₂. The organic layer was dried over MgSO₄ (anhydrous),filtered, concentrated on the rotary evaporator and dried under highvacuum. The product was used in the next step without furtherpurification.

B) Condensation Reactions 1. Amide Formation

The N-acylated amino acid (1 eq), 1-Hydroxybenzotriazole (HOBt) (1.05eq) and HMBA hydrochloride (1.05 eq) were placed in a round bottom flaskequipped with a stirring bar and fitted with a rubber septum. Dry CH₂Cl₂(4 mL/mmol) and NEt₃ (1.05 eq) were added under positive N₂ pressure viaa syringe through the rubber septum. The flask was immersed in an icebath, and the reaction mixture was stirred. After sufficient chilling,1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.05 eq) was addedin one portion, and the reaction mixture was allowed to stir to roomtemperature overnight. TLC (96:4, CH₂Cl₂/MeOH, v/v) revealed completionof reaction. The reaction mixture was diluted with CH₂Cl₂ and washedwith 1N HCl, H₂O and saturated NaCl. The organic phase was dried overMgSO₄ (anhydrous), filtered and concentrated on the rotary evaporator.The residue was dried under high vacuum, and the crude product waspurified by column chromatography on silica gel eluting with 9:1,CH₂Cl₂/acetone, v/v. NDH 4486: Mp=135-136° C.; R_(f)=0.54 (9:1,CH₂Cl₂/acetone); Yield=68%. Exact mass (ESI) calculated for C₂₄H₃₇N₂O₅[M+H] 433.2697. found 433.2676.

2. Ester Formation

The preparation of esters was carried out as described for amides withthe exception of replacing HOBt with 0.2 eq of DMAP. TLC analysis wasperformed using 7:3, hexanes/EtOAC, v/v while chromatographicpurification was carried out using 8:2, hexanes/EtOAC, v/v.

1. NDH 4638: The crude material was purified by column chromatographyeluting with 7:3, hexanes/EtOAc. R_(f)=0.27 (7:3, hexanes/EtOAc);Yield=47%. Exact mass (ESI) calculated for C₂₇H₄₀NO₆ [M+H] 474.2850.found 474.2878.

2. NDH 4642: The crude material was purified by column chromatographyeluting with 7:3, hexanes/EtOAc. R_(f)=0.32 (7:3, hexanes/EtOAc);Yield=61%. Exact mass (ESI) calculated for C₃₂H₄₈NO₆ [M+H] 542.3476.found 542.3494.

3. NDH 4639: The reaction solution was concentrated, and the residuetaken up in EtOAc. The organic layer was extracted with a small amountof water, saturated NaHCO₃, water, and finally saturated NaCl. The crudeproduct was purified twice by column chromatography-first eluting with8:2, hexanes/EtOAc and then 94:6, CH₂Cl₂/Et₂O. R_(f)=0.36 (8:2,hexanes/EtOAc); Yield=24%. Exact mass (ESI) calculated for C₃₃H₅₂NO₆[M+H] 558.3789. found 558.3809.

4. NDH 4647: The reaction solution was concentrated, and the residuetaken up in EtOAc. The organic layer was extracted with a small amountof water, saturated NaHCO₃, water, and finally saturated NaCl. The crudematerial was purified by column chromatography eluting with 8:2,hexanes/EtOAc. R_(f)=0.70 (8:2, hexanes/EtOAc); Yield=43%. Exact mass(ESI) calculated for C₂₆H₄₀NO₄ [M+H] 430.2952. found 430.2968.

5. NDH 4648: The reaction solution was concentrated, and the residuetaken up in EtOAc. The organic layer was extracted with a small amountof water, saturated NaHCO₃, water, and finally saturated NaCl. The crudematerial was purified by column chromatography eluting with 8:2,hexanes/EtOAc. R_(f)=0.69 (8:2, hexanes/EtOAc); Yield=62%. Exact mass(ESI) calculated for C₃₁H₄₇NO₄Na [M+Na] 520.3397. found 520.3429.

6. NDH 4640: The reaction solution was concentrated, and the residuetaken up in EtOAc. The organic layer was extracted with a small amountof water, saturated NaHCO₃, water, and finally saturated NaCl. The crudematerial was purified by column chromatography eluting with 8:1:1(CH₂Cl₂/DIPE/hexanes). R_(f)=0.89 (8:1:1, CH₂Cl₂/DIPE/hexanes);Yield=57%. Exact mass (ESI) calculated for C₂₆H₃₈NO₅ [M+H]444.2744.found 444.2750.

7. NDH 4641: The reaction solution was concentrated, and the residuetaken up in EtOAc. The organic layer was extracted with a small amountof water, saturated NaHCO₃, water, and finally saturated NaCl. The crudematerial was purified by column chromatography eluting with 8:1:1(CH₂Cl₂/DIPE/hexanes). R_(f)=0.92 (8:1:1, CH₂Cl₂/DIPE/hexanes);Yield=64%. Exact mass (ESI) calculated for C₃₁H₄₆NO₅ [M+H] 512.3370.found 512.3391.

NMR Data 1) NDH 4486

¹HNMR (CDCl₃) δ6.83 (d, 1H, ³J=8.0 Hz, ArH), 6.76 (d, 1H, ⁴J=1.85 Hz,ArH), 6.72 (dd, 1H, ³J=8.1 Hz, ⁴J=1.85 Hz, ArH), 6.12 (bs, 1H, amideNH), 5.57 (s, 1H, ArOH), 5.30 (bs, 1H, O—CH₂CH═), 5.16 (d, 1H, ³J=7.4Hz, carbamate NH), 5.06 (m, 1H, (CH₃)₂—C═CH—), 4.60-4.50 (m, 2H,C(O)O—CH₂—), 4.41-4.29 (2×dd, 2H, ²J=14.5 Hz, ³J=5.5 Hz, Ar—CH₂—N), 3.93(dd, 1H, ³J_(NH)=8.7 Hz, ³J_(CH)=6.1 Hz, CO—CH), 3.85 (s, 3H, Ar—O—CH₃),2.22-2.10 (m, 1H, CH—(CH₃)₂), 2.10-2.08 (m, 4H, ═C—CH₂—CH₂—C═),1.69-1.63 (m, 6H, ═(CH₃)₂), 1.58 (s, 3H, CH₃—C═), 0.95 (d, 3H, ³J=6.8Hz, CH(CH ₃)—CH₃) and 0.90 (d, 3H, ³J=6.8 Hz, CH(CH₃)—CH ₃). Exact mass(ESI) Calculated for C₂₄H₃₇N₂O₅ [M+1]433.2697. found 433.2676.

2) NDH 4631

¹HNMR (methanol-d₄) δ7.16-7.10 (bd, 2H, 2×ArH-3), 7.04-6.98 (m, 2H,2×ArH-4), 6.89-6.84 (bd, 2H, 2×Ar-6), 3.11-2.99, (m, 8H, CH ₂NCH₂CH₂CH₂CH ₂NCH ₂), 2.90- 2.81 (m, 2H, 2×CH(CH₃)₂), 2.14 (bs, 6H,2×ArCH₃), 1.97-1.89 (m, 4H, 2×NCH₂CH ₂CH₂N), 1.80-1.72 (m, 4H, NCH₂CH₂CH ₂CH₂N), and 1.21 (bd, 12H, ³J=6.95 Hz, 2×HC(CH ₃)₂). Note: Theprotons 2×OCNHCH ₂ are masked beneath the methanol-d₄ CH₃ peak centeredat δ3.30.

3) NDH 4638

¹HNMR (CDCl₃) δ6.97 (d, 1H, ³J=8.05 Hz, ArH-6), 6.75 (d, 1H, ⁴J=1.8 Hz,ArH-3), 6.71 (dd, 1H, ³J=8.05 Hz, ⁴J=1.8 Hz, ArH-5), 5.60 (d1H, ³J=9.05Hz, NH), 4.90 (bd, 1H, ³J=9.55 Hz, bornyl H-2), 4.34 (dd, 1H, ³J=8.9 Hz,⁴J=4.5 Hz, CO—CH), 3.77 (s, 3H, ArOCH₃), 2.85 (t, 2H, ³J=7.5 Hz, ArCH₂CH₂CO), 2.73 (t, 2H, ³J=7.45 Hz, ArCH₂CH ₂CO), 2.41-2.36 (m, 1H, bornylH-3exo), 2.27-2.20 (m, 1H, (CH₃)₂CH), 2.13 (s, 3H, COCH₃), 1.94-1.89 (m,1H, bornyl H-6 endo), 1.78-1.72 (m, 1H, bornyl H-5 exo), 1.68 (t, 1H,J=4.40 Hz, bornyl H-4), 1.37-1.28 (m, 1H, bornyl H-6 exo), 1.25-1.16 (m,1H, bornyl H-5 endo), 1.02 (d, 3H, ³J=6.85 Hz, CH ₃(CH₃)CH—), 0.99-0.94(m, 4H, bornyl H-3 endo and CH₃(CH ₃)CH—), 0.89 (s, 3H, one bornyl C-7CH₃), 0.87 (s, 3H, one bornyl C-7 CH₃) and 0.84 (s, 3H, bornyl C-1 CH₃).

4) NDH 4639

¹HNMR (CDCl₃) δ6.97 (d, 1H, ³J=8.05 Hz, ArH-6), 6.75 (d, 1H, ⁴J=1.8 Hz,ArH-3), 6.71 (dd, 1H, ³J=8.10 Hz, ⁴J=1.8 Hz, ArH-5), 5.60 (d, 1H,³J=8.95 Hz, NH), 4.91 (bd, 1H, ³J=9.60 Hz, bornyl H-2), 4.34 (dd, 1H,³J=8.95 Hz, ⁴J=4.55 Hz, CHCO), 2.84 (t, 2H, ³J=7.58 Hz, ArCH₂—), 2.69(t, 2H, ³J=7.58 Hz, ArCH₂CH ₂CO—), 2.41-2.34 (m, 3H, bornyl H-3 exo andArCH₂CH ₂COCH ₂—), 2.27-2.20 (m, 1H, (CH₃)₂CH—), 1.94-1.89 (m, 1H,bornyl H-6 endo), 1.78-1.72 (m, 1H, bornyl H-5 exo), 1.68 (t, 1H,³J=4.42 Hz, bornyl H-4), 1.58-1.50 (m, —COCH₂CH ₂(CH₂)₄CH₃ maskedbeneath D₂O peak), 1.36-1.17 (m, 10H, —COCH₂CH₂(CH ₂)₄CH₃, bornyl H-5endo and bornyl H-6 exo), 1.02 (d, 3H, ³J=6.85 Hz, CH ₃(CH₃)CH—),1.00-0.93 (m, 4H, CH₃(CH ₃)CH— and bornyl H-3 endo) 0.89 (s, 3H, onebornyl C-7 CH₃) and 0.86-0.83 (m, 9H, one bornyl C-7 CH₃, bornyl C-1 CH₃and —CO(CH₂)₆CH ₃).

5) NDH 4640

¹HNMR (CDCl₃) δ7.01 (d, 1H, ³J=7.75 Hz, ArH-6), 6.75 (d, 1H, ⁴J=1.6 Hz,ArH-3), 6.73 (d, 1H, ³J=8.05 Hz, ArH-5), 5.97-5.89 (m, 1H, ArCH₂CH═CH₂),5.61 (d, 1H, ³J=8.95 Hz, NH), 5.10-5.04 (m, 2H, ArCH₂CH═CH ₂), 4.92-4.89(m, 1H, bornyl H-2), 4.35 (dd, 1H, J_(NH)=8.95 Hz, J_(CH)=4.55 Hz,—CH(NH)CO—), 3.80 (s, 3H, ArOCH₃), 3.34 (d, 2H, J=6.70 Hz, ArCH₂CH═CH₂), 2.42-2.34 (m, 1H, bornyl H-3 exo), 1.95-1.89 (m, 1H, bornylH-6 endo), 1.78-1.71 (m, 1H, bornyl H-5 exo), 1.68 (t, 1H, J=4.45 Hz,bornyl H-4), 1.37-1.28 (m, 1H, bornyl H-6 exo), 1.25-1.16 (m, 1H, bornylH-5 endo), 1.03 (d, 3H, ³J=6.90 Hz, CH ₃(CH₃)CH—), 0.99-0.94 (m, 4H,bornyl H-3 endo and CH₃(CH ₃)CH—), 0.89 (s 3H, one bornyl C-7 CH₃), 0.86(s, 3H, one bornyl C-7 CH₃) and 0.84 (s, 3H, bornyl C-1 CH₃).

6) NDH 4641

¹HNMR (CDCl₃) δ7.01 (d, 1H, ³J=8.0 Hz, ArH-6), 6.74 (d, 1H, ⁴J=1.65 Hz,ArH-3), 6.72 (dd, 1H, ³J=8.0 Hz, ⁴J=1.8 Hz, ArH-5), 5.96-5.88 (m, 1H,ArCH₂CH═CH₂), 5.60 (d, 1H, ³J=9.1 Hz, NH), 5.35 (bt, 1H, J=7.15 Hz,—OCH₂CH═C—), 5.13-5.03 (m, 4H, ArCH₂CH═CH ₂ and 2 vinyl H of farnesylchain), 4.72-4.61 (m, 2H, —OCH ₂CH═C—), 4.33 (dd, 1H, J_(NH)=9.15 Hz,J_(CH)=4.6 Hz, (CH₃)₂CHCHCO), 2.25-2.17 (m, 1H, (CH₃)₂CH—), 2.13-1.93(m, 8H, 4 allylic —CH₂— of farnesyl chain), 1.70 (s, 3H, —OCH₂C═C(CH₃)—), 1.66 (s, 3H, center CH ₃ of farnesyl chain), 1.58 (s, 6H, —C═C(CH₃)₂), 0.996 (d, 3H, ³J=6.85 Hz, CH ₃(CH₃)CH—) and 0.917 (d, 3H, ³J=6.90Hz, CH₃(CH ₃)CH—).

7) NDH 4642

¹HNMR (CDCl₃) δ6.97 (d, 1H, ³J=8.0 Hz, ArH-6), 6.75 (d, 1H, ⁴J=1.85 Hz,ArH-3), 6.70 (dd, 1H, ³J=8.05 Hz, ⁴J=1.85 Hz, ArH-5), 5.60 (d, 1H,³J=9.1 Hz, NH), 5.34 (m, 1H, —OCH₂CH═C—), 5.12-5.04 (m, 2H, 2 vinyl H offarnesyl chain), 4.72-4.60 (m, 2H, —OCH ₂CH═C—), 4.33 (dd, 1H,J_(NH)=9.15 Hz, J_(CH)=4.6 Hz, (CH₃)₂CHCHCO), 3.79 (s, 3H, ArOCH₃), 2.84(t, 2H, ³J=7.5 Hz, ArCH₂—), 2.73 (t, 2H, ³J=7.5 Hz, ArCH₂CH ₂CO—),2.26-2.17 (m, 1H, (CH₃)₂CH—), 2.12 (s, 3H, —COCH₃), 2.11-1.93 (m, 8H, 4allylic —CH₂— of farnesyl chain), 1.70 (2, 3H, —OCH₂CH═C(CH ₃)—), 1.66(s, 3H, center CH ₃ of farnesyl chain), 1.58 (s, 6H, —C═C(CH ₃)₂), 0.99(d, 3H, ³J=6.8 Hz, CH ₃(CH₃)CH—) and 0.91 (d, 3H, ³J=6.90 Hz, CH₃(CH₃)CH—).

8) NDH 4647

¹HNMR (CDCl₃) δ7.10 (d, 1H, ³J=7.75 Hz, ArH-6), 6.97 (dd, 1H, ³J=7.70Hz, ⁴J=1.55 Hz, ArH-5), 6.91 (s, 1H, ArH-3), 5.54 (d, 1H, ³J=8.95 Hz,NH), 4.97-4.87 (m, 1H, bornyl H-2), 4.37 (dd, 1H, ³J=9.05 Hz and 4.45Hz, COCH), 2.84 (septet, 1H, ³J=6.96 Hz, CH(CH₃)₂), 2.44-2.35 (m, 1H,bornyl H-3 exo), 2.30-2.21 (m, 1H, (CH₃)₂CHCH(NH)CO), 2.16 (s, 3H,ArCH₃), 1.96-1.88 (m, 1H, bornyl H-6 endo), 1.80-1.72 (m, 1H, bornyl H-5exo), 1.71-1.67 ((bt, 1H, ³J=4.40 Hz, bornyl H-4), 1.37-1.29 (m, 1H,bornyl H-6 exo), 1.22-1.18 (m, 7H, bornyl H-5endo and ArCH(CH ₃)₂), 1.03(d, 3H, ³J=6.85 Hz, CH ₃(CH₃)CHCH(NH)CO), 1.01-0.93 (m, 1H, bornyl H-3endo), 0.95 (d, 3H, ³J=6.95 Hz, CH₃(CH ₃)CHCH(NH)CO), 0.89 (s, 3H,bornyl C-7 CH₃), 0.87 (s, 3H, bornyl C-7 CH₃) and 0.84 (s, 3H, bornylC-1 CH₃).

9) NDH 4648

¹HNMR (CDCl₃) δ7.09 (d, 1H, ³J=7.75 Hz, ArH-2), 6.97 (dd, 1H, ³J=7.75Hz, ⁴J=1.45 Hz, ArH-3), 6.90 (s, 1H, ArH-5), 5.54 (d, 1H, ³J=9.15 Hz,NH), 5.35 (bt, 1H, OCH₂—CH═), 5.12-5.04 (m, 2H, 2 vinyl protons),4.75-4.61 (m, 2H, OCH₂—CH═), 4.38-4.32 (dd, 1H, ³J=9.18 Hz and ³J=4.58Hz, CH—CO), 2.89-2.86 (septet, 1H, ³J=6.86 Hz, ArCH(CH₃)₂), 2.28-2.18(m, 1H, (CH₃)₂CHCH(NH)CO), 2.15 (s, 3H, ArCH₃), 2.14-1.93 (m, 8H, 4 CH₂units of fornesyl moiety), 1.71 (s, 3H, O—CH₂CH═C(CH ₃)—), 1.66 (s, 3H,CH₂CH₂C═C(CH ₃)CH₂—), 1.58 (s, 6H, C═C(CH ₃)₂), 1.20 (d, 6H, ³J=6.95 Hz,ArCH(CH ₃)₂), 1.00 (d, 3H, ³J=6.85 Hz, CH ₃(CH₃)CHCH(NH)CO) and 0.92 (d,3H, ³J=6.9 Hz, CH₃(CH ₃)CHCH(NH)CO).

Examples of Aspect II

Synergism of anti-inflammatory responses by anti-inflammatory agentscovalently coupled to amino acids (Aspect II) was demonstrated bypreparation of the S-naproxen-valine conjugate, and screening it in theMEVM against CEES challenge. MEVM is a standard in vivo assay forassessment of anti-inflammatory potential in addressingchemically-induced injury to rodent skin. CEES is one of theinflammation inducers employed in the MEVM assay. A compound of theinvention, Formula (IV-acid) (NDH 4476) provided four times betterinflammation suppression (44%) than naproxen itself under the sameconditions. The corresponding ethyl ester analog (IV-ethyl ester) (NDH4535) was equipotent but the 3.3-dimethylbutyl ester(IV-3,3-dimethylbutyl-) (NDH 4596) was superior at 52% inflammationsuppression. The latter molecule also was an inhibitor of AChEdisplaying anti-cholinergic activity with an IC₅₀ of 18.6 μM.

The phenylalanine conjugate of S-naproxen (esterified as the3,3-dimethylbutyl ester) shown in Formula (V) (NDH 4572) displayed animpressive 83% suppression of CEES-induced inflammation while S-naproxenitself yielded a mere 11% suppression of CEES inflammation. Thesix-carbon ester not only adds lipophilicity and promotes solubility ofthe NSAID-amino acid pharmaceutical in ointment excipients, but throughits action as a bioisostere of choline it provides anticholinergicactivity. For a discussion of how anticholinergic activity canfacilitate anti-inflammatory responses see S. C. Young et al,Investigation of anticholinergic and non-steroidal anti-inflammatoryprodrugs which reduce chemically-induced skin inflammation, J. Appl.Tox., 2012, 32: 135-141. The choline bioisostere 3,3-dimethylbutylalcohol provides cholinesterase inhibition in the finalanti-inflammatory drug-amino acid-choline bioisostere construct. For thenaproxen-phenylalanine platform, Formula (V), (also known as NDH 4572)this choline mimic generates an IC₅₀ value of 4.7 M against AChE.

The phenylalanine conjugate of the NSAID diclofenac (esterified as the3,3-dimethylbutyl ester; Formula (VI)) (NDH 4578) displayed a complete(100%) suppression of induced inflammation in the mouse. In the sameassay diclofenac itself displayed a mere 17% suppression ofinflammation.

Despite the fact that it is an NSAID, topical ibuprofen by itself wasfound to be a dermal irritant, adding 11% additional inflammation toCEES-induced injury. Furthermore, vanillylamine is only a weakanti-inflammatory; however, the triple conjugate of ibuprofen,vanillylamine, and valine, Formula (VII) (NDH 4479), provided a 94%suppression of CEES-induced inflammation.

Aspect II: Design and Synthesis of the NSAID-Amino Acid Conjugates andNSAID-Amino Acid-Anticholinergic Conjugates

The NSAID-amino acid conjugates (as esters or as free carboxylic acids)were synthesized by the following general method. All NSAIDs employedherein bear a pendant carboxylic acid group. To illustrate how suchmolecules are linked to the amino acid carrier the designation NSAID-CO—is used to convey that the fundamental ring system of the NSAID isattached through its carboxyl moiety. The required amino acids (0.60mmol) were first esterified with ethyl alcohol, n-butyl alcohol, or3,3-dimethylbutyl alcohol in toluene with p-toluenesulfonic acid as acatalyst. The amino acid esters could be isolated, crystallized, andpurified in 55-85% yields if so desired. Then the requisite NSAID (0.60mmol), and HOBt (0.66 mmol) were added in CH₂Cl₂ (5 mL) under a nitrogenatmosphere. The reaction contents were stirred at room temperature for15 min, until the solution became clear. EDC-HCl (1.1 equiv., 126 mg,0.66 mmol) was then added and the reaction contents were stirred at roomtemperature overnight (16 hr). Distilled water was added and the organiclayer was separated. The aqueous phase was then extracted with methylenechloride (25 mL) and the two organic layers were combined and washedwith 1 M HCl (2×50 mL), saturated NaHCO₃ (50 mL), and brine. The organiclayer was then dried over anhydrous MgSO₄, filtered, and concentrated toyield the final product, which was purified via column chromatographyusing a gradient separation with hexanes (100 to 50%) and ethyl acetate(0 to 50%) as the eluting solvent mixture.

Yields on the amide-forming step were 89-99% and after columnchromatography were homogeneous by TLC. These NSAID-amino acid-esterconjugates were sufficiently pure for in vitro (AChE) screening or invivo (MEVM) testing. Hydrolysis of these esters in 1:1 water:THF with 1mmol of Na₂CO₃ could free the carboxylic acids (giving the simpleNSAID-amino acid conjugate if so desired) in 40% yield. Products wereidentified by exact mass spectrometry with experimental values within+/−0.02 amu of the theoretical mass. In this fashion, on the valineplatform, (IV-ethyl ester, NDH 4535) (white solid, mp 135-139° C.) and(IV-3,3-dimethylbutyl ester, NDH 4596) (clear oil R_(f)=0.30 with 4:1hexane:ethyl acetate) and (IV-free acid, NDH 4476) (white solid,164-166° C.) were prepared. While this method is suitable for anyNSAID-amino acid or NSAID-amino acid ester, the specific productsprepared by this route were NDH 4651, NDH 4652, NDH 4653, and NDH 4654.Scheme I illustrates this pathway with any alcohol (R′—OH) and anycarboxyl-bearing NSAID but the method has been specifically applied tothese alcohols: ethanol, n-butanol, 3,3-dimethylbutyl alcohol,2-(trimethylsilyl)ethyl alcohol, and to these NSAIDs: ibuprofen,naproxen, indomethacin, and diclofenac.

For the proline conjugates, two structurally-related products wereobserved via nuclear magnetic resonance (NMR) spectroscopy, evenfollowing extensive chromatographic purification. In all cases, thepercentage of the second product ranged from 13 to 19%, depending on theNSAID. The final products were homogeneous by TLC. It was determinedthat the sterically hindered proline amide bond undergoes cis-transisomerization (Scheme II) which can be detected via NMR (vide infra).Cis-trans isomerization of the proline peptide bond is well documentedand plays an important role in protein folding.

Aspect II: Design and Syntheses of Amino Acid Conjugates RequiringSpecialized Transformations

A. Preparation of Amino Acid Conjugates which Include a Ketone Body(3-hydroxybutyrate) Illustrated with NDH 4571 as an Example

The labile 3-hydroxy group requires protection before it can be linkedto an amino acid platform. For this the TBDMS-protected 3-hydroxybutyricacid ((R)-3-[(tert-butyl)dimethylsilyloxy] butanoic acid) was firstprepared according to the procedure of D. Seebach, et. al. (HelveticaChimica Acta, 79(3), 670 (1996)) and used as the starting material.Seebach's protected acid compound was subsequently converted to thethiazolide of the silyl-protected butanoic acid, first structure shownin Scheme III. The protected acid (1.776 g, 8.133 mmol),mercaptothiazoline (970 mg 8.133 mmol), andN,N′-dicyclohexylcarbodiimide (DCC) (1.762 g, 1.05×8.133 mmol) weredissolved in 40 mL of CH₂Cl₂. The flask was immersed in an ice bath, andafter sufficient chilling, a catalytic amount of 4-dimethylaminopyridine(DMAP) was added. The ice bath was removed stirring for 2 h, and themixture was stirred at room temperature for an additional 2 h. The ureawas filtered off, and the filtrate extracted with saturated NaHCO₃, 1NHCl and saturated NaCl. The organic layer was dried over MgSO₄, filteredand concentrated. A portion of the crude (850 mg) was purified by columnchromatography on silica gel (70 g) eluting with hexanes/ethyl acetate.8:2 to give a 78% yield of a bright yellow oil, R_(f)=0.49.

While Step 1 can employ any of the anti-inflammatory amino acids, thepathway is illustrated with L-valine. The thiazolide (331.9 mg, 1.038mmol), L-valine (128 mg, 1.05×1.038 mmol) and diisopropylethylamine (268mg, 362 μL, 2×1.038 mmol) were dissolved in a mixture of 5.2 mL each ofwater and THF. The reaction mixture was stirred vigorously overnight.The colorless mixture was diluted with CH₂Cl₂ and extracted with 1 NHCl. The organic layer was concentrated, and the residue was dissolvedin ether. The ether solution was extracted with saturated NaHCO₃. Thebicarbonate layer was extracted twice with ether and then carefullyacidified to pH=1 using 4N HCl. The resulting aqueous mixture wasextracted with CH₂Cl₂. The organic layer was dried over MgSO₄, filtered,and concentrated. This product of Step 1 was used in Step 2 withoutfurther purification.

In Step 2, the N-substituted valine derivative (229.6 mg, 0.723 mmol),HOBt (103 mg, 1.05×0.723 mmol), 4-hydroxy-3-methoxybenzylaminehydrochloride, also known as vanillylamine hydrochloride, (144 mg,1.05×0.723 mmol) and NEt₃ (77 mg, 106 μL, 1.05×0.723 mmol) weredissolved in CH₂Cl₂ (7 mL). The solution was stirred and chilled in anice bath. To the cold mixture was added EDC (153 mg, 1.1×0.723 mmol).The mixture was allowed to stir to room temperature overnight. Themixture was diluted with CH₂Cl₂ and extracted with water, 1N HCl,saturated NaHCO₃, and saturated NaCl. The organic layer was dried overMg SO₄, filtered and concentrated. The crude product was purified bycolumn chromatography on silica gel (50 g) eluting with CH₂Cl₂/MeOH,94:6 (v/v), R_(f)=0.40, to give a 70% yield. Although Scheme III, Step 2shows the incorporation of vanillylamine, any nucleophilicanti-inflammatory could be used (e.g., a phenolic-protected vanillylalcohol).

In Step 3, the silyl-protected conjugate (229 mg, 0.506 mmol) wasdesilylated by dissolving in 5 mL of MeOH, adding NH₄F (94 mg, 5×0.506mmol) and heating at 60° C. for 7 days. The solution was cooled to roomtemperature and concentrated under reduced pressure. The crude productwas purified by column Chromatography on silica gel (40 g) eluting withCH₂Cl₂/MeOH, 98:2 (v/v) and increasing to 92:8 to give a yield of 84%,(R_(f)=0.23 (CH₂Cl₂/MeOH, 94:6 (v/v), mp=164-174° C. with rapidheating). Spectral evidence confirmed the structure of NDH 4571. ¹H NMR(acetone d₆) δ: 7.70-7.64 (m, 1H, —NH— of valine), 7.44 (d, 1H, ³J=4.5Hz, CH₃CH(OH)—), 6.90 (m, 1H, Ar), 6.74-6.69 (m, 2H, Ar), 4.31-4.25 (m,3H, —NCH(CH₃)₂— and Ar—CH ₂—), 4.09-4.03 (m, 1H, CH₃CH(OH)—), 3.80 (s,3H, Ar—OCH₃), 2.42-2.27 (m, 2H, —C(H(OH)CH ₂CO—), 2.20-2.07 (m, 1H,—CH(CH₃)₂), 1.14-1.11 (m, 3H, —CH(CH ₃)₂), 0.93 (t, 3H, ³J=6.80 Hz,CH(CH ₃)₂) and 0.91-0.88 (2 sets of doublets, 3H, ³J=6.85 Hz each, CH₃CH(OH)—).

B. Preparation of NSAID-Amino Acid Conjugates with Free Amino AcidCarboxyls (Illustrated with NDH 4476 or Compound IV-Acid)

While the amino acid conjugates of NSAIDs (those with a free amino acidcarboxyl) can be prepared by hydrolysis of the ester products of SchemeI, a far better route involves the thiazolide pathway. Thus, thesynthesis of IV-acid was carried out as described in step 1 for thesynthesis of NDH 4571 using the thiazolide of (S)-naproxen beingcondensed with L-valine to render a 68% yield of a white solid, NDH 4476or IV-acid. Mp=164-166° C.; R_(f)=0.56 (rocket), CH₂Cl₂/MeOH, 9:1 (v/v);Exact mass (ESI) Calculated for C₁₉H₂₄NO₄ [M+H] 330.1700. found330.1680. ¹H NMR (CDCl₃) δ: 7.72-7.76 (m, 3H, Ar), 7.36 (d, 1H, ³J=8.40Hz), 7.14 (dd, 1H, ³J=8.95 Hz, ⁴J=2.3 Hz), 7.10 (s, 1H), 5.82 (d, 1H,³J=8.35 Hz), 4.45-4.42 (m, 1H), 3.90 (s, 3H), 3.77 (q, 1H, ³J=7.15 Hz),2.16-2.09 (m, 1H), 1.60 (d, 3H, ³J=7.25 Hz), 0.87 (d, 3H, ³J=6.85 Hz)and 0.74 (d, 3H, ³J=6.85 Hz).

Although illustrated herein with S-naproxen and L-valine, thisthiazolide route can be used for any carboxyl-terminated NSAID and anyamino acid co-reactant.

C. Preparation of a Formula II Example Wherein and NSAID and a Vanilloidare Linked to an Amino Acid Through Nitrogen Atoms, Illustrated with NDH4479 (Compound VII)

Compound VII or NDH 4479 is one of the most potent anti-inflammatoriesobserved in the MEVM, with 110% suppression of phorbol-induced and 94%suppression of CEES-induced inflammation. The synthesis of VII wascarried out as described in steps 1 and 2 for the synthesis of NDH 4571but using the thiazolide of ibuprofen to give a 72% yield of a solid.Mp=56-66° C. with rapid heating; purification by column chromatographywith silica gel and CH₂Cl₂/acetone, 92:8 (v/v); R_(f)=0.23,CH₂Cl₂/acetone, 92:8 (v/v); Exact mass (ESI) Calculated for C₂₆H₃₇N₂O₄[M+H]441.2748. found 441.2742. ¹H NMR (CDCl₃) δ: 7.17-7.03 (m, 4H, Ar ofIbuprofen), 6.85-6.64 (m, 3H, Ar of vanillamine), 6.25-6.06 (m, 1H, NHof valine), 5.86-5.76 (m, 1H, NH of vanillamine), 5.57 (br s, 1H, ArOH),4.40-4.08 (m, 3H, —NCHCO— and Ar—CH₂—), 3.84-3.82 (m, 3H, ArOCH₃),3.59-3.49 (m, 1H, ArCH(CH₃)CO—), 2.45-2.40 (m, 2H, (CH₃)₂CHCH ₂—),2.12-1.95 (m, 1H, (CH₃)₂CHCH₂—), 1.85-1.76 (m, 1H, (CH₃)₂CHCH(NH)CO—),1.49-1.43 (m, 3H, ArCH(CH ₃)CO—) and 0.88=0.63 (m, 12H, (CH ₃)₂CHCH₂—and (CH ₃)₂CHCH(NH)CO—).

D. Alternative Preparation of NDH 4535

While the synthesis of NDH 4535 could be achieved as described in SchemeI with ethanol as the esterifying alcohol, a much higher yield can beachieved by the thiazolide route. The synthesis of NDH 4535 is bestcarried out as described in step 1 for the synthesis of NDH 4571 usingthe thiazolide of (S)-naproxen, L-valine ethyl ester hydrochloride andTHF only as solvent. The product was purified by column chromatographyon silica gel and eluting with hexanes/ethyl acetate, 7:3 (v/v) to yield84% of a crystalline product: mp=100-102° C., R_(f)=0.43 (hexanes/ethylacetate 7:3 (v/v)). Exact mass (ESI) Calculated for C₂₁H₂₈NO₄ [M+H]358.2013. found 358.2021. ¹H NMR (CDCl₃) δ: 7.73-7.68 (m, 3H, Ar), 7.38(dd, 1H, ³J=8.5 Hz, ⁴J=1.8 Hz, Ar), 7.13 (dd, 1H, ³J=8.9 Hz, ⁴J=2.55 Hz,Ar), 7.10 (d, 1H, ⁴J=2.45 Hz, Ar), 4.50-4.46 (m, 1H, N—CHCO), 4.14-4.01(m, 2H, OCH ₂CH₃), 2.10-2.03 (m, 1H, —CH(CH₃)₂), 1.60 (d, 3H, ³J=7.2 Hz,—CH(CH ₃)CO—), 1.15 (t, 3H, ³J=7.15 Hz, OCH₂CH ₃), 0.85 (d, 3H, ³J=6.85Hz, —CH(CH ₃)₂) and 0.74 (d, 3H, ³J=6.85 Hz, —CH(CH ₃)₂).

E. Preparation of a Mixed Vanilloid-Amino Acid Platform Illustrated withNDH 4483

Since both vanillylamine and vanillyl alcohol possess anti-inflammatoryactivities and in similar fashion to the amino acid valine, the triplecombination consistently displays MEVM numbers >65%. The synthesis ofNDH 4483 was carried out as described in steps 1 and 2 for the synthesisof NDH 4571 but using the thiazolide carbamate of4-acetoxy-3-methoxyvanillyl alcohol. The product was purified by columnchromatography on silica gel and eluting with CH₂Cl₂/MeOH, 94:6 (v/v) togive a 61% yield of a white solid: R_(f)=0.53 (CH₂Cl₂/MeOH, 92:8 (v/v).Exact mass (ESI) Calculated for C₂₄H₃₁N₂O₄ [M+H] 475.2075. found475.2058.

¹H NMR (CDCl₃) δ: 6.97 (d, 1H, ³J=8.0 Hz, H-5 of vanillyl alcohol), 6.92(s, 1H, H-2 of vanillyl alcohol), 6.88 (d, 1H, ³J=8.2 Hz, H-6 ofvanillyl alcohol), 6.82 (d, 1H, ³J=8.0 Hz, H-5 of vanillylamine), 6.75(s, 1H, H-2 of vanillylamine), 6.72 (d, 1H, ³J=7.9 Hz, H-6 ofvanillylamine), 5.61 (s, 1H, ArOH), 5.34 (d, 1H, ³J=8.5 Hz, NH ofvaline), 5.05-4.99 (m, 2H, ArCH₂O—), 4.41-4.27 (m, 2H, ArCH ₂NH—),3.96-3.91 (m, 1H, —NHCHCO—), 3.82 (s, 3H, ArOCH₃), 3.80 (s, 3H, ArOCH₃),2.29 (s, 3H, ArOCOCH ₃), 2.13 (m, 1H, —CH(CH₃)₂), 0.97 (d, 3H, ³J=6.8Hz, —CH(CH₃)CH ₃) and 0.91 (d, 3H, ³J=6.8 Hz, —CH(CH ₃)CH₃).

The amino acid-3,3-dimethylbutyl esters lacking the NSAID moiety wereall inactive in inhibition of AChE as were the NSAID-amino acid ethyland n-butyl esters. These displayed IC₅₀ values greater than 100 M andprecise IC₅₀ values could not be determined due to solubilitylimitations of the compound being tested. Some of these simpleconjugates did, however, possessed modest (usually 5-44%)anti-inflammatory activity in the mouse ear vesicant model (e.g.,IV-acid and IV-ethyl ester at 40-44% and the n-butyl esters designatedNDH 4651-4654 at <25%). These data indicate that the choline mimicsalone (or AA linked choline mimics) do not have a high affinity forAChE. Low micromolar anticholinesterase IC₅₀ activities are obtainedonly when the choline mimics are covalently linked to an aromatic andlipophilic NSAID such as diclofenac. While the relationship between theIC₅₀ values for inhibition of AChE and the measured anti-inflammatoryeffects in the MEVM is not linear, it can be observed (Table I) thatcompounds with the lowest IC₅₀'s (e.g., below 3.3 micromolar) displayedsuperior inflammation suppression percentages for at least one of theinflammation-inducers. (See NDH 4537, 4577, 4578, and 4591)

TABLE I NSAID-Amino Acid - 3,3-dimethylbutyl Esters (other structuralexamples are described elsewhere herein) NDH AChE % % # NSAID Amino AcidIC₅₀ (μM) CEES^(a) TPA^(a) 4618 Naproxen Proline >100*  34 Irritant 4619Ibuprofen Proline NT 24 35  4617 Indomethacin Proline >25* 25  68***4628 Diclofenac Proline 15.4 +/− 0.1 10  76*** 4614 Ibuprofen Glycine27.9 +/− 2.7 Irritant 18  4613 Naproxen Glycine NT  66** 54** 4615Indomethacin Glycine 6.63 +/− 0.4 21 55** 4627 Diclofenac Glycine >50*Irritant 45** 4576 Ibuprofen Phenyl- 4.34 +/− 0.2 Irritant Irritantalanine 4572 Naproxen Phenyl- 4.77 +/− 0.2  83** 42** alanine 4577Indomethacin Phenyl- 2.55 +/− 0.7  62** 79** alanine 4578 DiclofenacPhenyl- 1.31 +/− 0.1  120** 90** alanine 4595 Ibuprofen Valine 8.91 +/−0.4 47 Irritant 4596 Naproxen Valine 18.6 +/− 3.0 51 22  4537Indomethacin Valine 3.29 +/− 0.3 59 107*** 4591 Diclofenac Valine 1.85+/− 0.1  85** 31  *A precise IC₅₀ could not be determined due to limitsin inhibitor solubility NT means not tested ^(a)Values differ from apositive control based on one-way ANOVA, **P < 0.05, ***P < 0.005

Representative Physical Data for Anti-Inflammatories of Aspect IIContaining Amino Acid Linkers

Stability. If vanillyl amine (i.e., 3-methoxy-4-hydroxybenzyl-NH—) isattached to any of these anti-inflammatory amino acid platforms itconstitutes a shelf-stable, slowly metabolized moiety. However, ifvanillyl alcohol (i.e., 3-methoxy-4-hydroxybenzyl-O—) is attached, theresulting candidate pharmaceuticals are unstable unless thefree-phenolic hydroxyl is protected by acylation. Acetate is a preferredprotecting group and the derived products are suitable therapeuticcandidates.

(S)-3,3-Dimethylbutylpyrrolidine-2-carboxylate

Light yellow liquid, 85% yield; R_(f) 0.12 (Hexanes:ethyl acetate 1:1);¹H NMR (500 MHz, CDCl₃)=6 0.92 (s, 9H), 1.53-1.57 (t, 2H, J=7.15 Hz),1.70-1.76 (m, 2H), 1.79-1.84 (m, 1H), 270, 2.05-2.11 (m, 1H), 2.85-2.90(m, 1H), 3.03-3.08 (m, 1H), 3.69-3.72 (dd, 1H, J=5.70, 8.60 Hz),4.14-4.17 (dt, 2H, J=1.70, 3.70 Hz); ¹³C NMR (125 MHz, CDCl₃): 025.5,29.6, 29.7, 30.3, 41.8, 47.1, 59.9, 62.7, 175.6; HRMS (m/z): calc. forC₁₁H₂₁NO₂ 200.1645; meas. 200.1638.

(S)-3,3-Dimethylbutyl-1-(2-(4-isobutylphenyl)propanoyl)pyrrolidine-2-carboxylate

Clear liquid, 93% yield; R_(f) 0.74 (Hexanes:ethyl acetate 1:1);according to ¹H NMR, 19.2% of the cis isomer of the proline peptide bondis present: ¹H NMR trans isomer (500 MHz, CDCl₃): 0 0.85-0.89 (m, 6H),0.92 (s, 9H), 1.38-1.42 (q, 3H, J=10.9 Hz), 1.54-1.57 (t, 2H, J=7.55Hz), 1.69-1.90 (m, 4H), 1.93-2.02 (m, 1H), 2.38-2.42 (dd, 2H, J=2.55,7.18 Hz), 3.17-3.50 (m, 2H), 3.64-3.76 (m, 1H), 4.10-4.20 (m, 2H),4.39-4.49 (m, 1H), 7.02-7.08 (m, 2H), 7.13-7.19 (m, 2H); cis isomer:00.85-0.89 (m, 6H), 0.87 (s, 9H), 1.38-1.42 (q, 3H, J=10.9 Hz),1.47-1.50 (t, 2H, J=7.50 Hz), 1.69-1.90 (m, 4H), 2.05-2.15 (m, 1H),2.38-2.42 (dd, 2H, J=2.55, 7.18 Hz), 3.173.50 (m, 2H), 3.64-3.76 (m,1H), 4.10-4.15 (m, 1H), 4.21-4.25 (m, 1H), 4.39-4.53 (m, 1H), 7.027.08(m, 2H), 7.13-7.19 (m, 2H); ¹³C NMR trans isomer (125 MHz, CDCl₃):020.3, 22.4, 22.5, 24.9, 29.6, 29.8, 30.1, 41.6, 44.5, 45.1, 46.8, 59.2,62.7, 127.3, 129.4, 138.4, 140.0, 172.3, 172.6; cis isomer: 0 20.4,22.3, 22.5, 24.8, 29.0, 30.2, 31.2, 41.7, 44.6, 45.0, 46.6, 58.9, 62.8,127.0, 127.3, 129.5. 129.6, 172.8, 172.9; Calc. for C₂₄H₃₇NO₃.0.25H₂O(392.06): C, 73.53; H, 9.64; N, 3.57. Found: C, 73.86; H, 9.41; N, 3.47.

(S)-3,3-Dimethylbutyl-1-((S)-2-(6-methoxynaphthalen-2-yl)propanoyl)pyrrolidine-2-carboxylate

White solid, 73% yield; MP 111.5-112.5° C.; R_(f) 0.62 (Hexanes:ethylacetate 1:1); according to ¹H NMR, 13.4% of the cis isomer of theproline peptide bond is present: ¹H NMR, trans isomer (500 MHz, DMF):00.99 (s, 9H), 1.52-1.57 (m, 5H), 1.92-1.97 (m, 2H), 2.04-2.07 (m, 1H),2.332.36 (m, 1H), 3.35-3.39 (m, 1H), 3.87-3.92 (m, 1H), 4.07 (s, 3H),4.17-4.26 (m, 3H), 4.55-4.57 (dd, 1H, J=4.20, 8.60 Hz), 7.31-7.34 (dd,1H, J=2.50, 9.00 Hz), 7.50 (d, 1H, J=2.50 Hz), 7.61-7.64 (dd, 1H,J=1.75, 8.45 Hz), 7.94-7.98 (t, 3H, J=8.65 Hz); cis isomer: 0 1.12 (s,9H), 1.52-1.57 (m, 5H), 1.76-1.80 (t, 2H, J=7.25 Hz), 1.92-1.97 (m, 2H),1.91-1.97 (m, 1H), 2.19-2.23 (m, 1H), 3.55-3.60 (m, 1H), 3.87-3.92 (m,1H), 4.07 (5, 3H), 4.43-4.45 (m, 2H), 7.31-7.34 (dd, 1H, J=2.50, 9.00Hz), 7.50 (d, 1H, J=2.50 Hz), 7.61-7.64 (dd, 1H, J=1.75, 8.45 Hz), 7.91(bs, 1H), 7.94-7.98 (1, 2H, J=8.65 Hz); ¹³C NMR, trans isomer (125 MHz,DMF): 020.1, 22.4, 31.1, 24.9, 41.7, 44.0, 46.9, 55.2, 59.4, 62.1, 63.2,106.1, 118.9, 126.3, 126.9, 127.4, 129.3, 129.4, 133.9, 137.3, 157.9,172.0, 172.4; cis isomer: 0 20.1, 22.4, 31.1, 41.8, 44.3, 46.6, 55.2,59.2, 62.1, 63.2, 106.0, 119.1, 125.9, 126.3, 126.9, 127.6, 129.3,129.4, 134.0, 137.1, 157.9, 172.5, 172.8; Calc. for C₂₅H₃₃NO₄ (411.53):C, 72.96; H, 8.08; N, 3.40. Found: C, 73.22; H, 7.98; N, 3.47.

(S)-3,3-Dimethylbutyl-1-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetyl)pyrrolidine-2-carboxylate

Yellow oil, 96% yield; R_(f) 0.47 (Hexanes:ethyl acetate 1:1); accordingto ¹H NMR, 18.5% of the cis isomer of the proline peptide bond ispresent: ¹H NMR, trans isomer (500 MHz, CDCl₃): δ 0.88 (5, 9H),1.41-1.45 (1, 2H, J=7.55 Hz), 1.93-2.03 (m, 2H), 2.05-2.09 (m, 1H),2.18-2.20 (m, 1H), 2.36 (5, 3H), 3.62-3.71 (m, 2H), 3.70 (5, 2H), 3.77(5, 3H), 4.03-4.07 (m, 2H), 4.30-4.33 (dd, 1H, J=4.55, 8.60 Hz),6.63-6.66 (dd, 1H, J=2.50, 9.00 Hz), 6.94-6.97 (m, 1H), 6.99 (d, 1H,J=2.50 Hz), 7.52 (d, 2H, J=8.45 Hz), 7.61-7.64 (m, 2H); cis isomer: δ0.89 (5, 9H), 1.47-1.51 (1, 2H, J=7.40 Hz), 1.84-1.90 (m, 1H), 1.93-2.03(m, 2H), 2.21 (5, 3H), 3.45-3.49 (m, 3H), 3.70 (5, 2H), 3.77 (s, 3H),4.08-4.13 (m, 2H), 4.58-4.61 (dd, 1H, J=1.95, 8.60 Hz), 6.63-6.66 (dd,1H, J=2.50, 9.00 Hz), 6.94-6.97 (m, 1H), 6.99 (d, 1H, J=2.50 Hz), 7.52(d, 2H, J=8.45 Hz), 7.61-7.64 (m, 2H); ¹³C NMR, trans isomer (500 MHz,CDCl₃): 0 13.6, 25.0, 29.5, 29.6, 29.7, 31.2, 41.6, 47.3, 55.7, 59.3,62.9, 101.7, 111.6, 112.9, 114.8, 129.1, 130.8, 130.9, 131.2, 134.0,135.6, 139.2, 156.0, 168.3, 168.8, 172.3; cis isomer: δ 13.5, 22.3,25.0, 29.1, 29.7, 31.7, 41.7, 46.8, 53.5, 59.6, 63.5, 101.6, 111.7,112.9, 114.8, 129.1, 130.8, 130.9, 131.2, 134.0, 135.6, 139.2, 156.1,168.3, 168.9, 172.3; Calc. for C₃₀H₃₅ClN₂O₅.0.5CH₂Cl₂ (581.53): C,63.00; H, 6.24; N, 4.82. Found: C, 63.34; H, 5.69; N, 4.81.

(S)-3,3-Dimethylbutyl-1-(2-(2-(2,6-dichlorophenylamino)phenyl)acetyl)pyrrolidine-2-carboxylate

Clear oil, 82% yield; R_(f) 0.31 (Hexanes:ethyl acetate, 4:1); accordingto ¹H NMR, 22.1% of the cis isomer of the proline peptide bond ispresent: ¹H NMR, trans isomer (500 MHz, CDCl₃): δ 0.86 (s, 9H),1.41-1.45 (1, 2H, J=15.0 Hz), 1.99-2.01 (m, 2H), 2.05-2.11 (m, 1H),2.11-2.17 (m, 1H), 3.62-3.71 (m, 2H), 3.72-3.87 (m, 3H), 4.06-4.14 (m,2H), 4.48-4.51 (dd, 1H, J=3.50, 8.60 Hz), 6.48 (d, 1H, J=7.75 Hz),6.84-6.89 (1, 1H, J=7.25 Hz), 6.91-6.94 (1, 1H, J=8.00 Hz), 7.06 (d, 1H,J=7.40 Hz), 7.15 (d, 1H, J=7.50 Hz), 7.29 (d, 2H, J=8.00 Hz); ¹H NMR,cis isomer (500 MHz, CDCl₃): δ 0.90 (s, 9H), 1.52-1.56 (1, 2H, J=7.45Hz), 1.88-1.94 (m, 2H), 2.13-2.19 (m, 1H), 2.23-2.32 (m, 1H), 3.54-3.62(m, 2H), 3.72-3.87 (m, 3H), 4.18-4.28 (m, 2H), 4.63-4.66 (dd, 1H,J=2.55, 8-0.53 Hz), 6.49-6.51 (m, 1H), 6.85-6.88 (m, 1H), 6.91-6.95 (1,1H, J=8.00 Hz), 7.04-7.08 (m, 2H), 7.30 (d, 2H, J=8.00 Hz); ¹³C NMR,trans isomer (125 MHz, CDCl₃): δ 24.9, 29.2, 29.6, 29.7, 39.2, 41.5,47.6, 60.1, 62.9, 117.8, 121.2, 123.8, 124.5, 127.6, 128.8, 130.0,130.7, 138.1, 143.7, 170.2, 172.2; ¹³C NMR, cis isomer (125 MHz, CDCl₃):δ 22.6, 29.6, 29.7, 31.6, 39.1, 41.7, 46.9, 60.1, 63.7, 117.8, 121.2,123.8, 124.7, 127.7, 128.8, 129.9, 130.6, 138.1, 143.7, 170.8, 172.3.

3,3-Dimethylbutyl 2-aminoacetate

Light yellow liquid, 58% yield; R_(f) 0.55 (Methylene chloride:methanol,9:1 with 3 drops NH₄OH); ¹H NMR (500 MHz, CDCl₃): δ 0.91 (s, 9H),1.42-1.47 (bs, 2H), 1.52-1.56 (1, 2H, J=7.50 Hz), 3.38 (s, 2H),4.13-4.18 (1, 2H, J=7.45 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 29.7, 29.8,41.8, 44.1, 62.7, 174.3; HRMS (m/z): calc. for C₈H₁₇NO₂ [M+1]: 160.1332;meas. 160.1321.

3,3-Dimethylbutyl 2-(2-(4-isobutylphenyl)propanamido)acetate

Clear liquid, 91% yield; R_(f) 0.37 (Hexanes:ethyl acetate 4:1); ¹H NMR(500 MHz, CDCl₃): δ 0.88 (d, 6H, J=6.60 Hz), 0.89 (s, 9H), 1.51 (d, 3H,J=7.15 Hz), 1.49-1.54 (1, 2H, J=5.80 Hz), 1.801.86 (m, 1H), 2.43 (d, 2H,J=7.20 Hz), 3.55-3.60 (q, 1H, J=7.15 Hz), 3.87-4.00 (dq, 2H, J=5.00,18.5 Hz), 4.12-4.16 (1, 2H, J=7.50 Hz), 5.83 (bs, 1H), 7.10 (d, 2H,J=8.00 Hz), 7.19 (d, 2H, J=8.00 Hz); ¹³C NMR (125 MHz, CDCl₃): 8 18.4,22.4, 29.5, 29.7, 30.2, 41.6, 44.9, 45.0, 46.6, 63.2, 127.4, 129.7,138.1, 140.9, 170.0, 174.6; Calc. for C₂₁H₃₃NO₃.0.25H₂O (351.99): C,71.66; H, 9.59; N, 3.98. Found: C, 71.84; H, 9.35; N, 4.02.

(S)-3,3-Dimethylbutyl 2-(2-(6-methoxynaphthalen-2-yl)propanamido)acetate

Clear oil, 99% yield; R_(f) 0.20 (Hexanes:ethyl acetate 4:1); ¹H NMR(500 MHz, CDCl₃): δ 0.87 (s, 9H), 1.45-1.49 (1, 2H, J=7.50 Hz), 1.59 (d,3H, J=7.20 Hz), 3.71-3.77 (q, 1H, J=7.15 Hz), 3.87-4.00 (dq, 2H, J=5.40,18.4 Hz), 3.90 (s, 3H), 4.09-4.14 (1, 2H, J=7.40 Hz), 5.85 (bs, 1H),7.10 (d, 1H, J=2.45 Hz), 7.12-7.15 (dd, 1H, J=2.55, 8.88 Hz), 7.36-7.39(dd, 1H, J=1.70, 8.43 Hz), 7.67 (s, 1H), 7.68-7.73 (1, 2H, J=8.55 Hz);¹³C NMR (125 MHz, CDCl₃): δ 18.4, 29.5, 29.7, 41.5, 41.6, 46.8, 55.3,63.2, 105.7, 119.2, 126.2, 126.3, 127.6, 129.0, 129.3, 133.8, 136.0,157.8, 169.9, 174.5; Calc. for C₂₂H₂₉NO₄ (371.47): C, 71.13; H, 7.87; N,3.77. Found: C, 70.97; H, 7.69; N, 3.80.

3,3-Dimethylbutyl2-(2-(1-(4-chlorobenzoyl}-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)acetate

Yellow solid, 89% yield; MP 118.5-120° C.; R_(f) 0.11 (Hexanes:ethylacetate 4:1); ¹H NMR (500 MHz, CDCl₃): δ 0.89 (s, 9H), 1.48-1.51 (1, 2H,J=7.55 Hz), 2.36 (s, 3H), 3.67 (s, 2H), 3.82 (s, 3H), 3.95 (d, 2H,J=5.40 Hz), 4.11-4.15 (1, 2H, J=7.50 Hz), 6.07-6.09 (1, 1H, J=5.00 Hz),6.68-6.71 (dd, 1H, J=2.55, 8.95 Hz), 6.91 (s, 1H), 6.90-6.94 (d, 1H,J=1-0.2 Hz), 7.45-7.48 (m, 2H), 7.64-7.67 (m, 2H); ¹³C NMR (125 MHz,CDCl₃): δ 13.4, 29.5, 29.7, 32.0, 41.5, 41.6, 55.8, 63.3, 100.8, 112.5,112.5, 115.1, 129.2, 130.2, 131.0, 131.3, 133.6, 136.4, 139.5, 156.3,168.3, 169.7, 170.2; Calc. for C₂₇H₃₁CIN₂O₅(499.00): C, 64.99; H, 6.26;N, 5.61. Found: C, 64.63; H, 5.94; N, 5.50.

3,3-Dimethylbutyl 2-(2-(2-(2,6-dichlorophenylamino)phenyl)acetamido)acetate

White solid, 70% yield; mp 118-119° C.; R_(f) 0.36 (Hexanes:ethylacetate 4:1); ¹H NMR (500 MHz, CDCl₃): δ 0.89 (s, 9H), 1.49-1.53 (1, 2H,J=7.50 Hz), 3.72 (s, 2H), 4.01 (d, 2H, J=5.05 Hz), 4.15-4.18 (1, 2H,J=7.45 Hz), 6.42-6.48 (bs, 1H), 6.49 (d, 1H, J=8.05 Hz), 6.88-6.92 (1,1H, J=7.40 Hz), 6.93-6.97 (1, 1H, J=8.15 Hz), 7.07-7.11 (1, 1H, J=7.85Hz), 7.17 (d, 1H, J=7.40 Hz), 7.31 (d, 3H, J=8.10 Hz); ¹³C NMR (125 MHz,CDCl₃): δ 29.1, 29.2, 40.2, 41.1, 41.3, 62.9, 117.2, 121.2, 123.7,124.0, 127.6, 128.4, 129.5, 130.2, 137.2, 142.5, 169.4, 171.3; Calc. forC₂₂H₂₆ClN₂O₃ (437.36): C, 60.42; H, 5.99; N, 6.41. Found: C, 60.36; H,6.09; N, 6.26.

(S)-3,3-Dimethylbutyl-2-amino-3-phenylpropanoate

Light yellow liquid, 36% yield; R_(f) 0.43 (Methylenechloride:hexanes:ethanol, 90:8:2); ¹H NMR (500 MHz, CDCl₃): δ 0.91 (s,9H), 1.42-1.45 (bs, 2H), 1.49-1.52 (1, 2H, J=7.70 Hz), 2.80-3.08 (dd,1H, J=7.95, 128 Hz), 2.83-3.06 (dd, 1H, J=7.95, 102 Hz), 3.65-3.70 (dd,1H, J=5.30, 7.93 Hz), 4.11-4.16 (m, 2H), 7.15-7.19 (d, 2H, J=7.15 Hz),7.21-7.24 (m, 1H), 7.26-7.30 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 29.6,29.7, 41.2, 41.7, 56.0, 62.7, 126.8, 128.6, 129.3, 137.4, 175.1; HRMS(m/z): calc. for C₁₅H₂₃NO₂ 250.1802; meas. 250.1791.

(S)-3,3-Dimethylbutyl2-(2-(4-isobutylphenyl)propanamido)-3-phenylpropanoate

Clear oil, 73% yield; R_(f) 0.59 (Hexanes:ethyl acetate 4:1); ¹H N.MR(500 MHz, CDCl₃): δ 0.82-1.01 (m, 15H), 1.34-1.53 (m, 5H), 1.78-1.90 (m,1H), 2.42-2.50 (dd, 2H, J=7.20, 10.9 Hz), 2.94-2.97 (1, 1H, J=3.80 Hz),2.91-3.07 (m, 1H), 3.44-3.53 (m, 1R), 4.01-4.16 (m, 2H), 4.734.84 (m,1R), 5.71-5.74 (m, 1R), 6.74 (d, 1H, J=7.20 Hz), 6.90-6.93 (m, 1R),7.05-7.16 (m, 5R), 7.15-7.20 (m, 2R); ¹³C NMR (125 MHz, CDCl₃): δ 18.19,22.42, 29.52, 29.53, 29.65, 29.69, 30.20, 30.24, 37.72, 37.76, 41.55,41.60, 45.06, 45.08, 46.62, 46.72, 52.92, 53.15, 63.16, 63.20, 126.89,126.96, 127.40, 127.41, 128.37, 128.45, 129.24, 129.29, 129.60, 129.62,135.63, 135.85, 137.67, 138.27, 140.74, 171.40, 171.49, 173.59, 173.96;Calc. for C₂₅H₃₉NO₃ (437.61): C, 76.85; H, 8.98; N, 3.20. Found: C,76.90; H, 9.19; N, 3.17.

(S)-3,3-Dimethylbutyl2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-3-phenylpropanoate

Clear oil, 82% yield; R_(f) 0.42 (Hexanes:ethyl acetate 4:1); ¹H N.MR(500 MHz, CDCl₃): δ 0.83 (s, 9R), 1.36-1.40 (1, 2H, J=7.45 Hz), 1.57 (d,3H, J=7.25 Hz), 2.94-3.05 (dq, 2H, J=5.75, 13.8 Hz), 3.65-3.70 (q, 1H,J=7.20 Hz), 3.91 (s, 3R), 3.99-4.10 (m, 2R), 4.73-4.78 (m, 1R), 5.78 (d,1H, J=7.75 Hz), 6.83-6.86 (m, 2H), 7.02-7.06 (1, 2H, J=7.65 Hz),7.09-7.15 (m, 3R), 7.29-7.32 (dd, 1H, J=1.80, 8.50 Hz), 7.58 (s, 1R),7.66 (dd, 2H, J=4.05, 8.68 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 18.1, 29.5,29.6, 37.7, 41.5, 47.0, 53.1, 55.4, 63.2, 105.6, 119.1, 126.2, 126.4,126.9, 127.5, 128.4, 129.0, 129.2, 129.3, 133.8, 135.6, 135.7, 157.8,171.3, 173.9; Calc. for C₂₉H₃₅NO₄ (461.59): C, 75.46; H, 7.64; N, 3.03.Found: C, 75.03; H, 7.59; N, 3.03.

(S)-3,3-Dimethylbutyl2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)3-phenylpropanoate

Light yellow oil, 92% yield; R_(f) 0.21 (Hexanes:ethyl acetate 4:1); ¹HNMR (500 MHz, CDCl₃): δ 0.87 (s, 9R), 1.43-1.49 (1, 2H, J=7.50 Hz), 2.19(s, 3R), 2.94-3.03 (m, 2H), 3.55-3.63 (q, 2H, J=17.5, 18.6 Hz), 3.80 (s,3R), 4.06-4.15 (m, 2R), 4.78-4.82 (m, 1R), 5.97 (d, 1H, J=8.05 Hz),6.72-6.75 (dd, 1H, J=2.55, 9.03 Hz), 6.77 (d, 2H, J=7.15 Hz), 6.86 (d,1H, J=2.45 Hz), 6.997.03 (m, 3R), 7.06-7.12 (m, 1R), 7.42 (d, 2H, J=8.75Hz), 7.53 (d, 2H, J=7.75 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 13.3, 29.5,29.7, 32.1, 37.6, 41.6, 52.9, 55.8, 63.3, 100.7, 112.5, 112.6, 115.2,127.0, 128.4, 129.1, 129.2, 130.2, 131.0, 131.2, 133.7, 135.4, 136.0,139.4, 156.4, 168.2, 169.3, 171.1; Calc. for C₃₄H₃₇ClN₂O₅ (589.12): C,69.32; H, 6.33; N, 4.76. Found: C, 68.85; H, 6.13; N, 4.60.

(S)-3,3-Dimethylbutyl 2-(2-(2-(2,6-dichlorophenylamino)phenyl)acetamido)-3-phenylpropanoate

Light yellow oil, 92% yield; R_(f) 0.65 (Hexanes:ethyl acetate 4:1); ¹HNMR (500 MHz, CDCl₃): δ 0.89 (s, 9H), 1.45-1.50 (m, 2H), 3.04-3.13 (m,2H), 3.59-3.72 (q, 2H, J=14.4, 45.4 Hz), 4.064.18 (m, 2H), 4.83-4.87.(m, 1H), 6.13 (d, 1H, J=7.80 Hz), 6.50 (d, 1H, J=7.90 Hz), 6.89-6.98 (m,3H), 6.95-6.98 (1, 1H, J=8.00 Hz), 7.10 (d, 2H, J=7.40 Hz), 7.16-7.19(m, 3H), 7.32 (d, 2H, J=8.05 Hz), 7.36 (bs, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 29.5, 29.7, 37.7, 41.0, 41.6, 53.3, 63.4, 117.7, 121.6, 124.2,124.4, 127.1, 128.0, 128.5, 128.8, 129.4, 130.1, 130.6, 135.6, 137.7,143.0, 170.9, 171.3; Calc. for C₂₉H₃₂Cl₂N₂O₃.0.5H₂0 (536.49): C, 64.92;H, 6.20; N, 5.22. Found: C, 64.99; H, 5.78; N, 5.05.

(S)-3,3-Dimethylbutyl 2-amino-3-methylbutanoate

Light yellow liquid, 64% yield; R_(f) 0.16 (hexanes:ethyl acetate: 4:1);¹H NMR (500 MHz, CDCl₃): δ 0.87 (d, 3H, J=6.85 Hz), 0.92 (s, 9H), 0.95(d, 3H, J=6.90 Hz), 1.38-1.45 (bs, 2H), 1.53-1.57 (1, 2H, J=7.70 Hz),1.97-2.02 (m, 1H), 3.23 (d, 1H, J=4.95 Hz), 4.13-4.16 (1, 2H, J=7.35Hz); ¹³C NMR (125 MHz, CDCl₃): δ 17.2, 19.4, 29.6, 29.7, 32.1, 41.8,60.0, 62.5, 175.7; HRMS (m/z): calc. for C₁₁H₂₃NO₂ 202.1802; meas.202.1784.

(S)-3,3-Dimethylbutyl2-(2-(4-isobutylphenyl)propanamido)-3-methylbutanoate

Clear liquid, 92% yield; R_(f) 0.55 (Hexanes:ethyl acetate 4:1); ¹H NMR(500 MHz, CDCl₃): δ 0.64 (d, 1.5H, J=6.90 Hz), 0.71-0.75 (dd, 3H,J=6.85, 9.20 Hz), 0.83 (d, 1.5H, J=6.85 Hz), 0.85-0.87 (m, 6H), 0.89 (s,4.5H), 0.90 (s, 4.5H), 1.46-1.53 (m, 5H), 1.79-1.86 (m, 1H), 1.99-2.10(m, 1H), 2.43 (d, 2H, J=7.20 Hz), 3.53-3.57 (q, 0.5H, J=7.15 Hz),3.57-3.62 (q, 0.5H, J=7.30 Hz), 4.07-4.14 (m, 2H), 4.43-4.49 (m, 1H),5.70-5.78 (dd, 1H, J=8.80, 24.8 Hz), 7.09-7.12 (m, 2H), 7.17-7.22 (m,2H); ¹³C NMR. (125 MHz, CDCl₃): 0 17.3, 17.5, 18.1, 18.3, 18.9, 19.0,22.2. 22.3, 29.5, 29.6, 29.7, 30.2, 31.2, 31.3, 41.6, 41.7, 45.0, 45.1,46.8, 46.9, 56.8, 56.9, 62.9, 63.0, 127.3, 127.4, 129.7, 138.6, 140.8,140.9, 171.9, 172.1, 174.1, 174.4; Calc. for C₂₄H₃₉NO₃ (389.57): C,73.99; H, 10.09; N, 3.60. Found: C, 73.90; H, 10.50; N, 3.52.

(S)-3,3-Dimethylbutyl2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-3-methylbutanoate

Clear oil, 99% yield; R_(f) 0.30 (Hexanes:ethyl acetate 4:1); ¹H NMR(500 MHz, CDCl₃): δ 0.73 (d, 3H, J=6.90 Hz), 0:84 (s, 9H), 0.85 (d, 3H,J=7.00 Hz), 1.38-1.42 (1, 2H, J=7.65 Hz), 1.54 (s, 3H), 1.60 (d, 3H,J=7.20 Hz), 2.05-2.10 (m, 1H), 3.71-3.77 (m, 1H), 3.90 (s, 3H),4.02-4.06 (1, 2H, J=7.45 Hz), 4.46-4.49 (dd, 1H, J=4.75, 8.73 Hz),7.09-7.14 (m, 2H), 7.36-7.40 (dd, 1H, J=1.65, 8.48 Hz), 7.68 (s, 1H),7.69-7.72 (dd, 2H, J=5.50, 8.60 Hz); ¹³C NMR (125:MHz, CDCl₃): δ 17.7,18.5, 19.0, 20.8, 29.5, 29.6, 31.3, 41.6, 47.1, 55.3, 57.1, 62.9, 105.7,119.1, 126.2, 126.4, 127.5, 129.0, 129.3, 133.8, 135.9, 157.7, 174.2,186.2; Calc. for C₂₅H₃₅NO₄ (413.55): C, 72.61; H, 8.53; N, 3.39. Found:C, 72.62; H, 8.87; N, 3.29.

(S)-3,3-Dimethylbutyl2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)3-methylbutanoate

White solid, 93% yield; mp 119-120° C., R_(f) 0.16 (Hexanes:ethylacetate 4:1); ¹H NMR (500 MHz, CDCl₃): δ 0.69 (d, 3H, J=6.90 Hz), 0.83(d, 3H, J=6.85 Hz), 0.88 (s, 9H), 1.44-1.48 (1, 2H, J=7.55 Hz),2.05-2.10 (m, 1H), 2.35 (s, 3H), 3.65 (m, 2H), 3.80 (s, 3H), 4.08-4.11(1, 2H, J=7.50 Hz), 4.48-4.52 (dd, 1H, J=4.75, 8.83 Hz), 6.07 (d, 1H,J=8.80 Hz), 6.68-6.71 (dd, 1H, J=2.50, 9.00 Hz), 6.89 (d, 1H, J=2.45Hz), 6.94 (d, 1H, J=9.00 Hz), 7.44-7.48 (m, 2H), 7.63-7.66 (m, 2H); ¹³CNMR (125 MHz, CDCl₃): δ 13.4, 17.6, 19.0, 29.5, 29.7, 31.2, 32.3, 41.6,55.7, 57.1, 63.1, 100.6, 100.9, 112.6, 112.7, 115.2, 129.2, 130.2,131.0, 131.2, 133.7, 136.2, 139.5, 156.3, 169.7, 171.7; Calc. forC₃₀H₃₇ClN₂O₅ (541.08): C, 66.59; H, 6.89; N, 5.18. Found: C, 66.48; H,7.12; N, 5.10.

(S)-3,3-Dimethylbutyl2-(2-(2-(2,6-dichlorophenylamino)phenyl)acetamido)-3-methylbutanoate

Clear oil, 100% yield; R_(f) 0.54 (Hexanes:ethyl acetate 4:1); ¹H NMR(500 MHz, CDCl₃): δ 0.85 (d, 3H, J=6.90 Hz), 0.88 (d, 3H, J=6.85 Hz),0.90 (s, 9H), 1.49-1.53 (1, 2H, J=7.55 Hz), 2.11-2.15 (m, 1H), 3.72 (s,2H), 4.12-4.16 (m, 2H), 4.53-4.57 (dd, 1H, J=4.90, 8.83 Hz), 6.16 (d,1H, J=8.90 Hz), 6.50 (d, 1H, J=7.95 Hz), 6.89-6.92 (td, 1H, J=0.95, 7.45Hz), 6.93-6.97 (1, 1H, J=8.00 Hz), 7.07-7.11 (td, 1H, J=1.55, 9.18 Hz),7.16-7.19 (dd, 1H, J=1.35, 7.50 Hz), 7.31 (d, 2H, J=8.05 Hz), 7.36 (s,1H); ¹³C NMR (125 MHz, CDCl₃): 0 17.8, 18.9, 29.6, 29.7, 31.4, 41.0,41.7, 57.2, 63.1, 117.8, 121.6, 124.1, 124.8, 128.0, 128.8, 129.9,130.5, 137.8, 143.0, 171.4, 171.9; Calc. for C₂₅H₃₂Cl₂N₂O₃ (479.44): C,62.63; H, 6.73; N, 5.84. Found: C, 62.46; H, 6.48; N, 5.66.

TABLE II Representative amino acid anti-inflammatory conjugates ofAspect II prepared by methods indicated herein are shown as examples,without limitation, of the compositions claimed herein. NDH4476:

NDH4535:

NDH4479:

NDH4537:

NDH4571:

NDH4572:

NDH4576:

NDH4577:

NDH4578:

NDH4591:

NDH4595:

NDH4596:

NDH4613:

NDH4614:

NDH4615:

NDH4617:

NDH4618:

NDH4619:

NDH4627:

NDH4628:

NDH4651:

NDH4652:

NDH4653:

NDH4654:

NDH4483:

All references cited herein are incorporated herein by reference intheir entireties.

What is claimed is: 1-21. (canceled)
 22. An anti-inflammatory drug-aminoacid conjugate, comprising: (a) at least one anti-inflammatory compoundconjugated with (b) an augmenting moiety comprising an anti-inflammatoryamino acid selected from the group consisting of valine, nor-valine,leucine, iso-leucine, glycine, cysteine, proline and phenylalanine;wherein conjugation is via the nitrogen atom of the amino acid of saidaugmenting moiety; and wherein the anti-inflammatory activity of theconjugate is greater than the sum of its parts.
 23. (canceled)
 24. Theconjugate of claim 22, wherein said amino acid is selected from thegroup consisting of valine, glycine, proline and phenylalanine.
 25. Theconjugate of claim 22, wherein said augmenting moiety is an amino acidester of H—OCH₂CH₂C(CH₃)₃ or H—OCH₂CH₂Si(CH₃)₃, or an amino acid amideof H₂NCH₂CH₂C(CH₃)₃ or H₂NCH₂CH₂Si(CH₃)₃.
 26. The conjugate of claim 22,wherein said anti-inflammatory compound is selected from the groupconsisting of non-steroidal anti-inflammatory drugs (NSAIDs), vanilloidsand ketone bodies.
 27. The conjugate of claim 26, wherein said NSAID isselected from the group consisting of diclofenac, ibuprofen, naproxen,and indomethacin; wherein said vanilloid is selected from the groupconsisting of vanillyl alcohol, 3-methoxy-4-acetyloxybenzyl alcohol, andvanillylamine; and wherein said ketone body is selected from the groupconsisting of 3-hydroxybutyrate and homologues thereof.
 28. Ananti-inflammatory drug-amino acid conjugate having the structure ofFormula (I)AI—NH—CHR—C(═O)O-Q¹  Formula (I) wherein AI represents ananti-inflammatory drug moiety selected from the group consisting of anNSAID-CO— moiety, a vanillyl-CO— moiety and a 3-hydroxybutyroyl moiety;wherein R is selected from the group consisting of hydrogen, isopropyland benzyl; and wherein Q¹ is selected from the group consisting ofalkyl and heteroalkyl.
 29. An anti-inflammatory drug-amino acidconjugate having the structure of Formula (II)AI—NH—CHR—C(═O)—NH-Q²  Formula (II) wherein AI represents ananti-inflammatory drug moiety selected from the group consisting of anNSAID-CO— moiety, a vanillyl-CO— moiety and a 3-hydroxybutyroyl moiety;wherein R is selected from the group consisting of hydrogen, isopropyland benzyl; and Q² is —CH₂CH₂C(CH₃)₃, —CH₂CH₂Si(CH₃)₃ or3-methoxy-4-hydroxybenzyl.
 30. (canceled)
 31. The conjugate of claim 28,wherein Q¹ is —CH₂CH₂C(CH₃)₃ or —CH₂CH₂Si(CH₃)₃.
 32. (canceled)
 33. Amethod of increasing the activity of an anti-inflammatory drug,comprising conjugating said anti-inflammatory drug with an amino acidaugmenting moiety to provide an amino acid conjugate of claim
 28. 34-36.(canceled)
 37. The method of claim 33, wherein Q¹ is selected from thegroup consisting of —CH₂CH₂C(CH₃)₃ and —CH₂CH₂Si(CH₃)₃. 38-39.(canceled)
 40. The amino acid conjugate of claim 28, wherein for theNSAID-CO— moiety, the NSAID is selected from the group consisting ofdiclofenac, naproxen and indomethacin.
 41. The amino acid conjugate ofclaim 29, wherein for the NSAID-CO— moiety, the NSAID is selected fromthe group consisting of diclofenac, ibuprofen, naproxen andindomethacin.
 42. A method of increasing the activity of ananti-inflammatory drug, comprising conjugating said anti-inflammatorydrug with an amino acid augmenting moiety to provide an amino acidconjugate of claim
 29. 43. The method of claim 42, wherein Q² is—CH₂CH₂C(CH₃)₃ or —CH₂CH₂Si(CH₃)₃.
 44. An anti-inflammatory drug-aminoacid conjugate selected from the group consisting of:


45. An anti-inflammatory drug-amino acid conjugate of claim 44 selectedfrom the group consisting of:


46. The anti-inflammatory drug-amino acid conjugate of claim 44, whereinsaid conjugate is


47. The anti-inflammatory drug-amino acid conjugate of claim 44, whereinsaid conjugate is


48. The anti-inflammatory drug-amino acid conjugate of claim 44, whereinsaid conjugate is


49. The anti-inflammatory drug-amino acid conjugate of claim 44, whereinsaid conjugate is


50. The anti-inflammatory drug-amino acid conjugate of claim 22, whereinsaid augmenting moiety is a valine ester or amide.
 51. Theanti-inflammatory drug-amino acid conjugate of claim 22, wherein saidaugmenting moiety is a phenylalanine ester or amide.
 52. Theanti-inflammatory drug-amino acid conjugate of claim 22, wherein saidaugmenting moiety is a proline ester or amide.
 53. The anti-inflammatorydrug-amino acid conjugate of claim 22, comprising: (a) ananti-inflammatory compound conjugated with (b) an augmenting moietycomprising an amino acid ester or amide, wherein conjugation is via thenitrogen atom of the amino acid of said augmenting moiety; wherein saidamino acid ester or amide is selected from the group consisting ofesters and amides of valine, glycine, proline and phenylalanine, whereinsaid anti-inflammatory compound is selected from the group consisting of(1) the non-steroidal anti-inflammatory drugs diclofenac, ibuprofen,naproxen, and indomethacin; (2) the vanilliods vanillyl alcohol,3-methoxy-4-acetyloxybenzyl alcohol, and vanillylamine; and (3) theketone bodies 3-hydroxybutyrate and homologues thereof; and wherein theanti-inflammatory activity of the conjugate is greater than the sum ofits parts.
 54. The conjugate of claim 22, wherein said augmenting moietyis an amino acid ester or amide of a vanilloid.
 55. The conjugate ofclaim 54, wherein said vanilloid is selected from the group consistingof vanillyl alcohol, vanillyl amine and phenol-protected derivativesthereof.